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Am J Physiol Cell Physiol 294: C797-C809, 2008. First published January 2, 2008; doi:10.1152/ajpcell.00493.2007
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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Nonredundant function of secretory carrier membrane protein isoforms in dense core vesicle exocytosis

Haini Liao,1 Jie Zhang,1 Svetlana Shestopal,1 Gabor Szabo,2 Anna Castle,1 and David Castle1

1Department of Cell Biology and Cell and Developmental Biology Program and 2Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia

Submitted 18 October 2007 ; accepted in final form 1 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Five secretory carrier membrane proteins (SCAMP-1, -2, -3, -4, and -5) have been characterized in mammalian cells. Previously, SCAMP-1 and -2 have been implicated to function in exocytosis. RNA inhibitor-mediated deficiency of one or both of these SCAMPs interferes with dense core vesicle (DCV) exocytosis in neuroendocrine PC12 cells as detected by amperometry. Knockdowns of these SCAMPs each decreased the number and frequency of depolarization-induced exocytotic events. SCAMP-2 but not SCAMP-1 depletion also delayed the onset of exocytosis. Both knockdowns, however, altered fusion pore dynamics, increasing rapid pore closure and decreasing pore dilation. In contrast, knockdowns of SCAMP-3 and -5 only interfered with the frequency of fusion pore opening and did not affect the dynamics of newly opened pores. None of the knockdowns noticeably affected upstream events, including the distribution of DCVs near the plasma membrane and calcium signaling kinetics, although norepinephrine uptake/storage was moderately decreased by deficiency of SCAMP-1 and -5. Thus, SCAMP-1 and -2 are most closely linked to the final events of exocytosis. Other SCAMPs collaborate in regulating fusion sites, but the roles of individual isoforms appear at least partially distinct.

neuroendocrine secretion; membrane fusion; amperometry

SECRETORY CARRIER MEMBRANE PROTEINS (SCAMPs) are ubiquitously expressed integral membrane proteins with four transmembrane spans. The prevalence and broad distribution of SCAMPs in membranes of endocytic recycling compartments, secretory vesicles, the trans-Golgi network, and the plasma membrane raise the prospects that they participate in a variety of membrane trafficking events. These include sorting, the formation of vesicular carriers, exocytosis, and endocytosis. Presently, however, there is not a consistent picture of their roles and, particularly, whether different SCAMP isoforms have distinct functions. Whole organism knockouts of SCAMP-1 in the mouse and of the lone SCAMP isoform expressed in Caenorhabditis elegans resulted in little or no obvious phenotypic defects in neural function (1, 9). In contrast, other findings clearly implicated selected mammalian SCAMPs (SCAMP-1 or -2) in the regulation of late events in exocytosis in mast cells and neuroendocrine cells and also suggested a role in constitutive clathrin-mediated endocytosis (1, 8, 9, 14, 23, 24). Potentially, these discrepancies may reflect differences in the cell types in which functions were assayed and, for exocytosis in particular, the distinction between examining processes mediated by fast versus slow secretory events (14, 23, 24). More significantly, in most mammalian cell types where multiple SCAMP isoforms are expressed (5, 23, 31), it is unclear to what extent there is functional redundancy or collaboration among isoforms in supporting various trafficking events like exocytosis. This issue seems particularly interesting because the functions of distinct isoforms have never been compared side by side even though they are situated adjacent to one another in recycling membranes and in the cell surface (5).

In the present study, we used RNA inhibitor (RNAi)-mediated knockdown of SCAMPs in PC12 cells to test for effects on DCV exocytosis. Our primary interest was in comparing SCAMP-1 and -2 to determine whether deficiencies in these previously identified regulators had unique or comparable effects. We extended some of our analyses to SCAMP-3 and -5 because they are also detected on the plasma membrane. SCAMPs were knocked down and evaluated for effects on DCV function as determined by a real-time analysis of norepinephrine (NE) secretion using amperometry. In addition, we analyzed individual knockdowns for effects on the distribution of DCVs at the plasma membrane, calcium signaling in response to depolarization, and cellular uptake and storage of NE to account for any changes that might indirectly affect our amperometric analyses. As in our previous study (24), amperometry enabled us to monitor multiple parameters of the fusion process including overall kinetics and dynamics of fusion pore opening, closure, and dilation. Our findings confirm a close association of SCAMP-1 and -2 with fusion pore formation and stability but indicated, quite interestingly, that their roles are at least partially distinct. Unexpectedly, SCAMP-3 and -5 also seemed to influence the same fusion process, thereby implicating multiple SCAMPs as nonredundant contributors to a single trafficking step.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents Specific inhibitory RNA [short interfering RNA (siRNA)] duplexes were designed against the coding regions of SCAMPs: 5'-TCTCGCTCGTCATGTTTAA-3' of SCAMP-1, 5'-GCGGCGAATTTGCATGTAA-3' of SCAMP-2, 5'-CGGAGTGATAGTTCATTCAATTTCT-3' of SCAMP-3, and 5'-TAACCAAGCGCCTCTACTA-3' of SCAMP-5. Distinct siRNAs targeted to different regions of SCAMP-1, -2, and -5 were used to confirm the specificity of effects caused by depletion of SCAMPs; the target sequences included 5'-AGACAGTGAAGCTTATGTA-3' of SCAMP-1, 5'-GCATTCAGTGACTCTGTTT-3' of SCAMP-2, and 5'-AGCCATGTTTCTACCAAGA-3' of SCAMP-5. siRNAs and nonspecific RNAi (Dharmacon no. 1) used as controls were synthesized by Invitrogen (Carlsbad, CA).

Electroporator and electroporation supplies were from Amaxa (Gaithersburg, MD). [3H]NE was from Perkin-Elmer Life and Analytical Sciences (Boston, MA). Polyclonal SCAMP-1 antibody 1{alpha} (16), SCAMP-2 antibody 2{Omega} (42), SCAMP-3 antibody 3β (14), and monoclonal anti-SCAMP 7C12 (4) have been characterized previously. Anti-SCAMP-5 (5{Omega}) was raised against synthetic peptide (C)TQYSATPNYTYSN (Covance, Denver, PA). Peptide coupling to maleimide-activated keyhole limpet hemocyanin (Imject, Pierce Endogen, Rockford, IL) and affinity purification of antibody on immobilized peptide (Sulfolink, Pierce Endogen) were performed as previously described (16). Anti-{gamma}-adaptin was from BD Biosciences (San Diego, CA); Alexa 488 and 594-conjugated antibodies and neutra-avidin conjugates were from Molecular Probes (Eugene, OR).

Cell Transfection and Expression The rat pheochromocytoma PC12 cell line (kindly provided by Edwin Chapman, University of Wisconsin, Madison, WI) was used for knockdown experiments. PC12 cells were transfected by electroporation (Amaxa Nucleofector II electroporator, program no. U-29, cuvettes provided by the Amaxa Nucleofector Kit V from Amaxa Biosystems) using 1 µg siRNA or nonspecific RNAi per 2 x 106 cells in 100 µl of electroporation buffer (Amaxa Cell Line Nucleofector Solution V). Knockdowns were evaluated 72 h after electroporation. Immunofluorescence microscopy and Western blot analysis were used to evaluate the efficiency of transfection and extent of knockdown, respectively.

Preparation and Immunostaining of Plasma Membrane Lawns PC12 cells cultured on glass coverslips coated with 100 µg/ml poly-D lysine (23) were incubated in sonication buffer (120 mM K+-glutamate, 20 mM K+-acetate, 10 mM EGTA, 20 mM HEPES, 0.5 mg/ml Mg2+-ATP, and 0.5 mM DTT; pH 7.2) and subjected to a pulse of sonication, which efficiently sheared the cells, leaving lawns of plasma membranes attached to the coverslip (20, 23). After being rinsed, samples were fixed with 3% formaldehyde and stained with anti-SCAMP and secondary antibodies as previously described (23). The preparations were examined, digital images were collected using OpenLab software, and the density of fluorescent staining was detected and counted using the Image J program with the plug-in Particle Detector and Tracker (30). Data are means ± SE and were analyzed by one-way ANOVA followed by the Tukey test for significant differences.

Total Internal Reflection Fluorescence Microscopy and Analysis PC12 cells were plated on poly-L-lysine-coated optical dishes. Cells were cotransfected with neuropeptide Y (NPY)-enhanced green fluorescent protein (EGFP) and siRNA (either SCAMP-1, -2, or -5) using the Amaxa nucleofection system. Imaging was performed in Leibovitz's L15 medium (Invitrogen). Images were acquired on an Olympus IX-70 microscope equipped with a x60 (1.45 numerical aperture) objective. EGFP was excited by a 488-nm argon ion laser, and fluorescence was collected in the total internal reflection fluoresence (TIRF) mode using a Photometrics Coolsnap charge-coupled device camera (Roper Scientific).

To analyze TIRF microscopy (TIRF-M) data, docked vesicles were detected and counted using the Image J program with the plug-in Particle Detector and Tracker (30). To normalize parameters for particle detection between different cells, the area of each cell footprint was determined, and images were cropped to make the total area equal to 5x the cell area. Vesicles were detected using the following parameters: radius (related to vesicle size) = 3, cutoff (quality of Gaussian fit for vesicle identification) = 3, and percentage of total fluorescence intensity = 0.5%. Data are means ± SE and were analyzed by one-way ANOVA followed by the Tukey test for significant differences.

Analysis of Intracellular Free Calcium in Response to Cell Depolarization To monitor free calcium levels, PC12 cells grown on coverslips were loaded in DMEM for 30 min at 37°C with 10 µM fura-2FF (added from a 400x stock in DMSO). After being washed, coverslips were mounted in a holder in medium containing 142 mM NaCl, 4.2 mM KCl, 1 mM Na2HPO4, 0.7 mM MgCl2, 2 mM CaCl2, and 10 mM HEPES (pH 7.2). Imaging was carried out at room temperature using an inverted microscope (Zeiss LSM510 confocal) equipped with a x63 (1.4 numerical aperture) oil objective with two-photon excitation at 830 nm provided by a titanium sapphire laser. Cells were depolarized by local perifusion with high-potassium buffer (105 mM K+; see Amperometry and Data Analysis); signals were collected continuously for 30 s using the blue channel (435–485 nm). Recordings were made on 15–20 cells of each type of sample. The ratio of fura-2FF fluorescence during stimulation to that obtained before stimulation was used to calculate calcium concentration (28). Maximal fluorescence was taken as the initial fluorescence and minimal fluorescence was determined from cells treated 3 min in medium containing 10 µM ionomycin and 10 mM CaCl2 (minimum fluorescence/maximum fluorescence = 0.04). Analyses and calculations were based on signals derived from a maximal rectangular area within the cytoplasm.

[3H]NE Uptake PC12 cells cultured in 24-well plates were labeled with [3H]NE by supplementation of the growth medium with 0.5 µCi/ml [3H]NE and 0.5 mM sodium ascorbate for an overnight incubation (16 h) at 37°C. Afterward, cells were rinsed twice and incubated for 1 h with growth medium. Labeled cells were lysed with RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM Tris, and 5 mM EDTA; pH 7.5), and the radioactivity was counted. The level of [3H]NE uptake was normalized to the number of cells estimated from a duplicate unlabeled dish.

Amperometry and Data Analysis Amperometry recording of NE secretion was as previously described (24). Briefly, cells were stimulated by local perfusion (3 psi from an Eppendorf Transjector 5246) of K+ depolarizing medium (105 mM KCl, 45 mM NaCl, 1 mM NaH2PO4, 0.7 mM MgCl2, 2 mM CaCl2, and 10 mM HEPES; pH 7.3) for 8 s. Currents were recorded using 5-µm carbon fiber electrodes (ALA Scientific Instruments, Westbury, NY) lightly contacting the cell surface and at a potential of +660 mV. Signals were fed to an Axopatch 200 amplifier, low pass filtered at 1 kHz, and digitized at 3.3 kHz by a computer running Lab Man (a custom data-acquisition program developed in the laboratory of G. Szabo). Recordings were analyzed using computer programs provided by Meyer Jackson and Payne Chang (University of Wisconsin). Current signals with amplitudes exceeding five times the background noise [root mean square (RMS) {approx} 0.3–0.4 pA] were counted as exocytotic events, and the onset was identified by a current rise to 1 x the RMS noise above baseline. Once identified, events with peak amplitude ≥ 4 pA having sharp upward/downward deflections were designated as spikes, whereas smaller events with amplitudes comparable with those of the foot currents of spikes (~2 pA) were identified as stand alone feet (SAF) (24, 39) (see RESULTS). Because noise levels were increased in selected experiments (RMS {approx} 0.5–0.7 pA), some records were filtered with a moving-average filter program (provided by Zhijian Lu, University of Virginia) to reduce the RMS level to 0.3–0.4 pA. Filtered records were used only in analyzing the latency of exocytotic events and counting the number of SAF versus spikes. Large spikes with peak amplitudes > 10 pA were used for the analysis of the prespike foot (PSF), half-width, rise time, and decay time of spikes (15). Due to cell-to-cell variability in the secretory response, results were calculated as means for all cells in an experimental sample (>40 cells/group from multiple transfections for each knockdown sample). A sample of cells transfected with nonspecific RNAi was set as the control for each experiment to control for variability between experiments. All results are reported as means ± SE, and one-way ANOVA was used to evaluate statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Distributions of SCAMPs on the Cell Surface Functional studies have suggested that cell surface-associated SCAMP-2 regulates exocytosis in mast cells and PC12 cells (14, 23, 24). To estimate the relative abundance of SCAMP-1, -2, -3, and -5 on the plasma membrane, we followed our previously published protocols to prepare membrane lawns of PC12 cells cultured on glass coverslips by light sonication, stained them with isoform-specific anti-SCAMP antibodies, and evaluated them by immunofluorescence (5, 6). For each type of sample, we quantitated the membrane surface area and number of stained puncta on plasma membrane sheets and calculated a labeling density. As shown in Fig. 1A, the densities for all four SCAMPs were not significantly different from one another (P > 0.05).


Figure 1
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  		Fig. 1. Plasma membrane-associated secretory carrier membrane proteins (SCAMPs) and knockdown (KD) of SCAMPs in PC12 cells. A: comparison of the surface densities of SCAMP-1 (SC1), -2 (SC2), -3 (SC3), and -5 (SC5) as estimated from immunofluorescent staining of plasma membrane sheets with isoform-specific antibodies. Numbers of fluorescent foci and surface area were quantitated (number of sheets used for quantitation: SC1 = 24, SC2 = 22, SC3 = 24, and SC5 = 26) in each type of preparation using the Image J program. Values in the bar graph are means ± SE. B: lysates from PC12 cells transfected with nonspecific (NS) short interfering RNA (siRNA) and siRNAs targeted for SC1, SC2, SC3, SC5 as well as SC1 + 2 combined using Amaxa nucleofection were analyzed by Western blot 3 days after transfection. Lysates were also blotted with anti-{gamma}-adaptin antibody for evaluating the amount of lysates loaded. C: bar graph showing levels of SCAMP expression after KD. The SCAMP expression level in control samples [transfected with NS RNA inhibitor (RNAi)] was set as 1. Error bars are SDs derived from 3–4 experiments. D: anti-SCAMP immunostaining of cells transfected with either NS RNAi (control) or siRNA targeted for SCAMPs as in A. Scale bar = 20 µm. E: anti-SCAMP1 immunostaining on plasma membrane sheets of cells transfected with either NS RNAi (control) or siRNA targeted for SC1. Anti-Vamp2 was used to detect sheets in both types of preparations. Scale bar = 10 µm. F: cell surface densities of SC1, SC3, and SC5 in cells transfected with NS RNAi (control) or siRNA targeted for SC2 and SC1 + 2. Greater than 15 sheets were used for the quantitaion of each sample. Data are means ± SE.

 
Evaluation of the Efficiency of SCAMP Knockdowns We transfected PC12 cells with siRNAs to specifically decrease the expression of SCAMPs. As indicated above, SCAMP-1, -2, -3, and -5 were present at similar densities on the plasma membrane. The level of SCAMP-4 detected in PC12 cells, however, was quite low (unpublished observations); thus, its knockdown was not investigated. SCAMP knockdown was achieved as described in MATERIALS AND METHODS. Western blot analysis with isoform-specific anti-SCAMP antibodies indicated that the level of knockdown was consistently ~90% (Fig. 1, B and C) and that the knockdowns were specific to the targeted SCAMPs. The fraction of cells transfected was also evaluated by immunofluorescence using the same antibodies, and depletion was routinely detected in >90% of the cells (Fig. 1D). We also confirmed using immunostaining of plasma membrane lawns that reductions of individual SCAMPs noted at the cellular level involved decreased incidence on the plasma membrane as well (Fig. 1E; SCAMP1 is used as an example). For each SCAMP (SCAMP-1, -2, and -5), two siRNAs resulting in ~90% knockdown were identified. One siRNA was used to show the major phenotypes of single SCAMP knockdowns, and the other was used to confirm findings and reduce the possibility of off-target effects (see Suppl. Fig. 1 for data using second siRNAs).1

SCAMP Knockdowns Do Not Visibly Alter Either Localization of DCVs at the Plasma Membrane or the Incidence of Other Plasmalemmal SCAMPs As a part of our analysis, we checked for visible effects of SCAMP knockdown on the proximity of DCVs to the plasma membrane, particularly as a previous study (35) has shown that knockout of other components of the exocytotic machinery, e.g., Sec1/Munc18, redistributes DCVs in chromaffin cells. PC12 cells were transfected with NPY-EGFP to fluorescently label the contents of DCVs (34), and, after knockdown of individual SCAMPs, TIRF-M was used to examine the incidence of DCVs that could be detected close to the plasma membrane. Representative images showing the footprints of cells transfected with nonspecific RNAi (control) or RNAi that depleted ~90% of SCAMP-1, -2, or -5 are shown in Fig. 2A. The incidence of EGFP-labeled DCVs within the evanescent field was quantitated using the Image J program as indicated in MATERIALS AND METHODS and is reported in Fig. 2B. As can be seen from Fig. 2B, none of the SCAMP knockdowns caused a significant change in the density of DCVs detected (P > 0.05). Because the evanescent field extended to a depth of 150 nm under the TIRF-M conditions used, we were unable to distinguish whether there were differences in tethering/docking of DCVs among the different samples. However, the similar densities observed in all samples argue that any effects of SCAMP deficiency on secretion are unlikely to involve altered access of DCVs to the cell surface for tethering/docking. Morphological docking of DCVs was also judged qualitatively to be quite similar by transmission electron microscopy on images of embedded and sectioned cells (unpublished observations).


Figure 2
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  		Fig. 2. Effect of SCAMP KD on the number of vesicles docked on plasma membranes. A: typical total internal reflection fluorescence microscopy images of plasma membrane-docked vesicles in cells transfected with control (NS), SC1, SC2, and SC5 siRNA. Scale bar = 10 µm. B: vesicle density in cells transfected with control, SC1, SC2, and SC5 siRNAs. Numbers of footprints analyzed in each case were as follows: control, 38; SC1, 33; SC2, 35; and SC5, 36. Data are means ± SE.

 
We also considered the possibility that knockdown of one SCAMP isoform might lead to an altered incidence of other SCAMP isoforms at the cell surface. However, immunostaining of plasma membrane lawns indicated that when knockdown efficiently decreased the level of a particular SCAMP, it apparently did not result in significantly increased or decreased levels of other SCAMPs at the cell surface (Fig. 1F). Thus, as previously reported for knockout of SCAMP1 (9), we have no evidence of compensation or reduced stability among isoforms when one isoform is deficient.

Effects of SCAMP Knockdowns Analyzed by Amperometry (SCAMP-1 and -2) We used amperometry on NE-loaded cells to analyze the effect of knockdowns on DCV exocytosis. In each experiment, a sample of cells transfected with nonspecific RNAi was compared with knockdown samples to provide a control and to take into account any experiment to experiment variability. Cells were stimulated individually by depolarization using locally perfused KCl; responses were recorded as previously described (24). Initially, we focused on the SCAMPs previously implicated to function in exocytosis and thus examined knockdowns of SCAMP-1 and -2 individually or in combination. Examples of traces from the different samples are shown in Fig. 3A. For each of the knockdowns, the total number of exocytotic events appeared to decrease in relation to the control, indicating inhibition of exocytosis. Examination at higher resolution showed that the recordings included larger spikes as well as smaller events (<4 pA); many of the latter resembled SAF, resulting from transient opening-closure (flicker) of fusion pores without dilation, that were identified in earlier studies (24, 41). Many spikes in all recordings had a PSF in which the slower increase in current preceding the spike is thought to distinguish initial opening from subsequent dilation of fusion pores (Fig. 3A, right, inset 1). Interestingly, the SAF appeared enriched in recordings from cells in the knockdown samples; examples are shown in Fig. 3A, right, insets 2 and 3). Their mean amplitude was comparable with that of the PSF (~2 pA). Thus, they appear distinct from the much smaller (mean amplitude ~0.4 pA) and often more extended events characterized previously in a study of synaptotagmin mutants in DCV exocytosis (41) and may represent a different type of kiss-and-run event (39). We did not detected 0.4 pA events in our study.


Figure 3
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  		Fig. 3. A: representative 30-s recordings of cells transfected with NS siRNA (control) and siRNAs targeted to individual and paired SCAMPs (SC1 KD, SC2 KD, and SC1+2 KD). Depolarization (105 mM K+; 8 s) was at time 0. Recordings were filtered at 500 Hz (8-pole Bessel) to reduce the noise. Inset 1 shows an example of a prespike foot (PSF) associated with many of the spikes and analyzed in all samples. Insets 2 and 3 show examples of events that were diagnosed as stand alone feet (SAF). B: analysis of exocytotic events (>300 exocytotic events) supporting the diagnosis that small events represent SAF. Left, plot of quantal release as a function of event amplitude showing a distinct population of disproportionately small events of less than ~4 pA amplitude thought to signify partial content release. Middle, plot of half-width-to-total duration ratio versus amplitude. The increased ratio at less than ~4 pA shows that small events are disproportionately broadened, characteristic of SAF. Right, plot of event duration versus amplitude. R and P values derived from linear regression are included. The results show that event duration and amplitude are not correlated and argue against the possibility that small events simply reflect exocytosis at increased distance from the electrode. The analysis was performed on 2 sets of data from different control samples. The outcomes were the same, and results from only 1 of the 2 sets are shown.

 
To support the interpretation that the smaller events represented SAF and were distinct from the larger events (spikes), we selected >300 events at random from control samples and analyzed selected characteristics (Fig. 3B). First, a plot of quantal release from each event (log scale) versus the event's amplitude (Fig. 3B, left) showed that smaller events (less than ~4 pA, left side of the dashed line) release disproportionately less charge than larger events (greater than ~4 pA). Also, the distribution of the quantal release for the larger events was not correlated with event amplitude. In this analysis, 4 pA is not an absolute boundary but rather has been selected to illustrate the distinct trends of the two populations. The distinct character of the smaller events seemed to fit the nature of SAF, where NE release is restricted by transient fusion pore flickering rather than dilation. Second, we measured the half-widths (width at half-height) and full-widths (total event duration) and examined the half-width-to-full-width ratio as a function of peak amplitude (Fig. 3B, middle). This analysis was similar to one included in a recent study (39) and a larger ratio is characteristic of SAF (e.g., Fig. 3A, right, insets 2 and 3) compared with the sharper, more triangular spikes (Fig. 3A, right, inset 1). Figure 3B shows that the smaller events were relatively broader than the large events; notably, the size distinction between the two populations corresponded closely to the boundary distinguishing "large kiss-and-run" events from spikes, as previously demonstrated (39). Thus, two independent measurements support the presence of distinct populations of small and large exocytotic events. Finally, we determined that duration was not correlated with amplitude for all events (Fig. 3B, right; correlation coefficient from linear regression analysis: ~0). This outcome effectively rules out the possibility that smaller and broader events simply reflect the diffusion of NE from release sites that are distant from the electrode because in this case event duration and amplitude should be inversely correlated. We performed this same set of analyses on an entirely separate set of data from other control samples and obtained results that were the same as those shown in Fig. 3B (not shown). Because cell shape was unchanged by knockdown (Fig. 1D) and electrode placement for recording was comparable for all samples, SAF recognized in knockdown samples should share the same properties as in control samples and also reflect transient opening (flickering) of fusion pores.

To assess the effects of knockdowns of SCAMP-1 and -2 quantitatively, we relied on previous strategies for characterizing the kinetics of fusion pore opening and dynamics and stability of initially opened fusion pores (24, 41).

Kinetics of fusion pore opening. Results from at least 40 cells of each type of sample were compiled and cumulative exocytotic events, including both spikes and SAF, averaged across all cells of one type, were plotted as a function of time (Fig. 4A, left). The initial slope of each curve was computed as the frequency of fusion pore opening. Knockdowns of SCAMP-1 and -2 each decreased the frequency compared with control, whereas the combined knockdown of both SCAMPs did not decrease frequency further (Fig. 4B). We also quantitated the total number of exocytotic events for each cell at 30 s after the initiation of depolarization and determined mean values (Fig. 4C). Knockdowns of SCAMP-1 or -2 had a similar inhibitory effect, and the combined knockdown caused an even greater decrease.


Figure 4
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  		Fig. 4. Effects of SCAMP KD on the kinetics of fusion pore opening. Data were obtained in 2 sets of experiments: SC1 KD and control 2 as well as SC2, SC1+2, and control 1. Both controls were the same NS RNAi. A: cumulative plots of total exocytotic events (spikes + SAF) over 30 s. Depolarization with 105 mM K+ was at time 0. Left, the number of events per cell was averaged from >40 cells under each condition. Right, expanded view of the first 2.5 s following depolarization clearly illustrating the lag in onset of release in the SC2 and SC1+2 KD samples. B: bar graph showing the frequency of fusion pore opening, which was derived from a linear fit of the initial part of the cumulative curve (2–10 s after the initiation of depolarization to avoid the delay phase). Each sample was paired with its corresponding control (NS RNAi). C: the maximum number of released vesicles was quantitated as the total number of exocytotic events at 30 s after the initiation of depolarization. D: bar graph presenting the average latency of the first exocytotic event after the initiation of depolarization. Each sample is shown with its paired control. All results from amperometry experiments are means ± SE. One-way ANOVA was used to evaluate statistical significance. **P < 0.001.

 
A delayed response of DCV exocytosis is typically detected in PC12 cells (3, 29) and may in part reflect the limited size of the ready-releasable pool of DCVs (15). We measured the average latency of the first event after depolarization (Fig. 4D). Quite interestingly, knockdowns of SCAMP-1 and -2 had different effects. The delay observed in the control was doubled by SCAMP-2 knockdown, whereas it remained unchanged by SCAMP-1 knockdown; in the combined SCAMP-1+ SCAMP-2 knockdown, it was not increased beyond the level observed for SCAMP-2 alone. These distinct effects are especially clear in expanded views of the initial portions of the cumulative event curves, as shown in Fig. 4A, right. Considered together, the frequency of DCV release events, the size of the release-competent vesicle pool, and the lag in response to stimulation reflect the regulatory steps upstream of exocytotic membrane fusion, e.g., docking, priming, and triggering of membrane fusion (25, 32). Our results using knockdowns suggest that some of the roles of SCAMP-1 and -2 within this series of events are quite similar; however, the lone effect of SCAMP-2 on the lag in response to stimulation argues that at certain levels these two SCAMPs are likely to have quite different roles.

Dynamics and stability of nascent fusion pores. While amperometry is unable to distinguish the different prefusion steps, it is particularly useful for examining the dynamics and stability of nascent fusion pores (39, 41, 43). Based on earlier studies (9, 24), we suspected that SCAMP-1 and -2 might have effects at this level. To begin to evaluate this possibility, we followed a previous strategy (24, 41) and plotted the number of SAF versus the number of spikes (reflecting the alternative outcomes of fusion pore closure and dilation) for each cell within a sample type and used the slope from a linear fit as an index of the likely fate of newly opened fusion pores (sample scatterplot; Fig. 5A). Notably, knockdowns of SCAMP-1 and particularly SCAMP-2 significantly increased the SAF-to-spike ratio, whereas the combined SCAMP-1 + SCAMP-2 knockdown did not increase the ratio beyond that attained with SCAMP-2 alone. Thus, when either SCAMP was deficient, the fusion pores tended to close rather than dilate, suggesting that the two SCAMPs might make similar contributions to fusion pore dynamics. The effects, however, did not appear additive when both SCAMPs were deficient. To gain insight into the stability of fusion pores in the newly opened state, we examined the duration of the PSF signal stemming from the limited efflux of NE. The distribution of PSF durations, which was measured from >100 spikes of each type of sample, could be fit by an exponential decay function (Fig. 5B, left) to obtain a mean lifetime ({tau}; Fig. 5B, right). As can be seen from the bar graph in Fig. 5B, knockdown of SCAMP-1 did not change {tau} relative to the control, whereas knockdown of SCAMP-2 caused a small decrease in {tau}, suggesting reduced stability of newly opened pores. Notably, combined deficiency of SCAMP-1 + SCAMP-2 significantly increased {tau}, implicating an increase in stability of nascent pores.


Figure 5
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  		Fig. 5. Effects of SCAMP KD on the dynamics and stability of nascent fusion pores. A: sample plot of SAF frequency versus spike frequency. Left, SC1+2 KD control. Each cell provides a single point. Right, the SAF-to-spike ratio was determined from the slope of the best-fit line, and values are shown in the bar graph. B: representative plot of the SC2 KD control. Left, density distribution of the PSF duration. The exponential fit (y = Ae–t/{tau}) is shown as a solid line. Right, bar graph showing mean lifetime ({tau}) values derived from curve fitting. Analysis was for spikes with amplitude of >10 pA and on >100 spikes in each case. The presentation of results and evaluation of statistical significance were performed as in Fig. 4. **P < 0.001.

 
We used the following model (3, 40) in our previous study (24) to analyze the kinetics of different states of the fusion pores:

Formula 1(1)
where C, O, and D represent closed, initially opened, and dilated fusion pores, respectively, and k values are rate constants for transition between states. Upstream steps, represented in aggregate by a rate constant of activation ka, are not resolved by amperometry; however, the following distal portion of the model is useful for analyzing events involving NE release:

Formula 2(2)

Accordingly, the SAF-to-spike ratio reflects kc/kd, and the mean {tau} of state O (initially opened fusion pores) equals 1/(kc + kd). For individual SCAMP-1 and -2 knockdowns, the SAF-to-spike ratio increased, but {tau} stayed the same or decreased mildly, indicating that kc + kd remained almost unchanged and that kc significantly increased (~38% and 90%, respectively), whereas kd decreased (~20% and 25%, respectively). For SCAMP-1 + SCAMP-2 knockdown, both {tau} and the SAF-to-spike ratio increased, indicating that kd decreased more significantly in the combined knockdown than in individual knockdowns (kd decreased ~50% and kc increased ~20%).

Quantal size of NE release events. For each spike, the integral of the current (spike area) is a measure of total charge (Q), which reflects the amount of NE released from a single vesicle. Substantially reduced NE release from each vesicle could result in misclassifying spikes as SAF and thereby artifactually increase the SAF-to-spike ratio. To consider this possibility, we determined the distributions of Q for exocytotic events in each type of sample. These distributions were compared by calculating the mean value of Q1/3 from Gaussian fits, which reiterates the distributions of DCV radii in PC12 cells (11). As shown in Fig. 6, all SCAMP knockdowns caused a similar slight decrease of Q1/3 compared with the control. Changes were not nearly sufficient to affect SAF and spike classifications. Therefore, this outcome indicates that the altered SAF-to-spike ratios observed for selected knockdowns reflected bona fide changes in nascent fusion pore dynamics and not altered NE quantal size.


Figure 6
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  		Fig. 6. Effects of SCAMP KD on the quantal size of catecholamine release. A: example of the distribution of the cube root of integrated spike areas [total charge (Q)1/3] for the SC2 control. The solid curved line is the Gaussian fit. B: mean values of Q1/3 for all types of samples are shown in the bar graph. Analysis was based on >500 spikes in each case. The presentation of results and statistical significance are as in Fig. 4. *P < 0.05; **P < 0.001.

 
We also measured parameters that are used to characterize the shape of amperometric spikes: rise time, half-width (the width at half-height), and decay time for the knockdown samples and corresponding controls. Individual knockdowns of SCAMP-1 and -2 did not cause changes in these parameters, and the only difference observed in the combined knockdown of SCAMP-1 + SCAMP-2 was an increased decay time (data not shown). This effect could reflect interference with fusion pore dilation and/or a decreased rate of dissociation of cathecholamine from the intragranular matrix (17, 36). Presently, we are unable to distinguish among these possibilities.

Amperometric Analysis of Knockdowns of SCAMP-3 and -5 While our main goal was to compare the roles of SCAMP-1 and -2, we were curious to distinguish whether other SCAMPs might also affect DCV exocytosis or whether there was selective involvement of SCAMP-1 and -2 in this process. Thus, we examined knockdowns of SCAMP-3 and -5. As for knockdowns of SCAMP-1 and -2, we constructed cumulative event curves and examined the frequency of fusion pore opening, the size of the releasable vesicle pool, and response latency as deduced from curve fitting. We also evaluated the SAF-to-spike ratio, PSF duration, spike shape, and quantal size. Deficiency of SCAMP-3 and -5 each decreased the frequency of fusion pore opening and size of the releasable pool to ~60–70% of the control, about the same as SCAMP-2 deficiency (Fig. 7, A and B). Neither knockdown affected the response latency (Fig. 7C). From these findings, we conclude that all four SCAMP isoforms are likely to contribute to regulating exocytotic steps that are proximal to fusion pore opening.


Figure 7
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  		Fig. 7. Effects of SC3 and SC5 KD analyzed by amperometry. All samples were compared with control cells (NS RNAi) that were examined in the same experiments. A–C: bar graphs showing the effects of KD on the frequency of fusion pore opening (A), maximum number of release events (B), and latency of response (C) as determined in Fig. 4. D and E: bar graphs showing the effects of KD on the SAF-to-spike ratio (D) and PSF duration (E) as determined in Fig. 5. F: bar graph showing the effects of KD on the quantal size of catecholamine release as determined in Fig. 6. Sample numbers analyzed were the same as in Figs. 4–6. The presentation of results and statistical significance are as in Fig. 4. *P < 0.05; **P < 0.001.

 
In knockdowns of SCAMP-3 and -5, there were no changes in the SAF-to-spike ratio compared with the control knockdown (Fig. 7D). Furthermore, an examination of the PSF duration indicated that {tau} was unchanged by deficiency in either of these SCAMPs (Fig. 7E). Thus, in contrast to SCAMP-1 and -2, the effects of deficiency in SCAMP-3 and -5 occurred upstream of fusion pore opening. With respect to the shape of amperometric spikes, the only changes observed were a decrease in half-width and decay time for SCAMP-3 knockdown. These may indicate a selective effect on NE diffusion through pores; however, the change was small, and we are presently hesitant to interpret it solely on the basis of amperometry. We also analyzed the quantal size of NE-release events in these knockdowns (Fig. 7F), and the small change observed for SCAMP-3 was not sufficient to affect SAF and spike classifications.

Effect of SCAMP Knockdowns on Depolarization-Induced Calcium Signaling We have used potassium-induced cell depolarization as a stimulus for DCV exocytosis in all of our assessments of SCAMP function by amperometry. An important issue to consider was whether any of the effects of knockdowns that were detected could potentially result from changes in calcium signaling linking depolarization to secretion. To explore this possibility, we loaded control or single-knockdown cells with fura2-FF, applied depolarization using locally perfused potassium, and continuously monitored fluorescence within the cytoplasm microscopically. As can be seen in Fig. 8, the profiles of calcium concentration change as a function of time were quite similar in SCAMP knockdowns compared with the control. Thus, we feel confident that the kinetics of calcium signaling are not affected by SCAMP knockdown. However, due to the resolution of the measurements, we cannot rule out the possibility that knockdown has a small effect on the magnitude of signaling. While this potentially could alter exocytosis indirectly, such an effect would likely be small relative to the extent of inhibition caused by knockdown. We also compared the calcium concentration profile to the instantaneous rates of DCV exocytosis obtained by differentiating the cumulative event plots shown in Fig. 4A. The curves were quite similar (not shown). Therefore, the maximal number of released vesicles shown in Figs. 4C and 7B approximated the pool of DCVs that were mobilized by depolarization and mainly contained DCVs that became primed for release; this number may somewhat underestimate the total pool available for release due to calcium return to resting levels.


Figure 8
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  		Fig. 8. Effect of SC1, SC2, and SC5 KD on intracellular calcium measured using fura 2-FF fluorescence in depolarized PC12 cells. A: sample calcium concentration profiles for cells treated with NS siRNA and SC1 siRNA. Data are plotted as means from 15–20 cells of each type. B: bar graph showing peak values of intracellular calcium in all samples normalized to cells treated with NS siRNA. For both A and B, error bars show SEM.

 
Levels of [3H]NE Uptake SCAMPs have been implicated not only in regulated exocytosis but also in endocytosis (8) and other trafficking processes (22, 27). Also, although we did not observe an appreciable effect of knockdown on quantal size of DCV exocytotic events, deficiencies in calcium-dependent activator protein for secretion (CAPS), another protein that regulates exocytosis, also interferes with NE loading of DCVs (33). For these reasons, we felt that it was important to investigate if depletion of SCAMPs altered either the formation of DCVs or their loading with NE, which was customarily done in amperometry experiments. When cellular NE uptake was measured by loading [3H]NE (Fig. 9), we found that knockdowns of SCAMP-1 and -5 decreased uptake by 23% and 49% of control, respectively, whereas knockdowns of SCAMP-2 and -3 were without effect. Thus, SCAMP-5 and, to a lesser extent, SCAMP-1 selectively affected NE uptake. These effects seem quite intriguing in light of the absence of strong effects of the same knockdowns on NE quantal size as measured by amperometry, and they raise the possibility that cellular uptake of NE may include another compartment in addition to those discharged by depolarization (see DISCUSSION).


Figure 9
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  		Fig. 9. Norepinephrine (NE) uptake measured in transfected PC12 cells. PC12 cells transfected with either NS siRNA (control) or SC1, SC2, SC3, SC5, and SC1 + 2 siRNAs were incubated with [3H]NE for 16–24 h, chased with fresh growth medium for 1 h after the removal of extracellular [3H]NE, and transferred to scintillation vials for the measurement of radioactivity. Levels of internalized [3H]NE were normalized for cell number estimated by cell counting using a duplicate unlabeled dish. Values for each sample are means ± SE. *P < 0.01.

 

   DISCUSSION
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In this study, we provided evidence that each of the four SCAMPs (SCAMP-1, -2, -3, and -5) that are abundant on the plasma membrane contributes to maintaining exocytotic sites so that fusion pore opening is expedited in response to a membrane-depolarizing stimulus. Upon depletion of any one of these isoforms, we observed decreases in the maximum number of vesicles released and frequency of fusion pore opening and, in the case of SCAMP-2, an increased response latency (Figs. 4 and 7). Among these isoforms, SCAMP-1 and -2 seem to be most intimately connected to fusion sites, especially as their depletion alters the behavior of newly opened fusion pores, enhancing their rapid closure and suppressing their tendency to dilate (Fig. 5). These findings confirm and extend previous observations that SCAMP-1 and -2 function in the late stages of exocytosis (9, 14, 23, 24). Overall, our evidence indicates that different SCAMP isoforms do not have fully redundant functions. Distinguishing roles of individual isoforms may reflect specific scaffolding functions for other exocytotic machinery and relative proximity to sites where apposed bilayers reorganize during the opening and development of fusion pores.

A General Role of SCAMPs on Priming of DCV Exocytosis? DCV exocytosis in neuroendocrine (endocrine) cells is divided into sequential stages of docking, priming, and calcium-triggered membrane fusion. In PC12 cells, a large fraction of DCVs morphologically appear close to or docked at the plasma membrane (Fig. 2 and unpublished electron micrographs). However, the kinetics of calcium-triggered exocytosis is much slower than in chromaffin cells (25, 26). Because PC12 cells mostly lack fusion-ready vesicles for fast release, the slow initial kinetics of exocytosis are thought to reflect mainly priming as the rate-limiting step (32). The frequency of fusion, size of the vesicle pool that is mobilized, and latency of fusion upon depolarization derived from measurements by amperometry (Figs. 4 and 7) should encompass the activities of sequential steps that lead to fusion pore opening at the plasma membrane, including docking and priming of DCVs. Also, compound exocytosis (18) and biogenesis of DCVs could potentially affect the experimental outcomes insofar as any defects might reduce the pool of releasable DCVs. Unfortunately, specific information about steps preceding fusion pore opening cannot be deduced from amperometry signals, and the lack of a fast fusion component in PC12 cells makes it impossible to distinguish the effects of SCAMP deficiency on the fast and/or slow components of exocytosis as observed in chromaffin cells (32). The reduced size of the releasable DCV pool resulting from SCAMP knockdowns (Figs. 4C and 7B) seems likely to reflect perturbation of mechanisms regulating the rate of exocytosis after depolarization. Because the distribution of DCVs at or near the cell surface appears unaffected by SCAMP knockdown (Fig. 2), we postulate that SCAMP deficiencies negatively affect the initiation of fusion pore opening mainly due to perturbations of DCV priming. Interestingly, depletion of SCAMP-2, either alone or in combination with other SCAMPs, specifically increased the latency of fusion in response to depolarization (Fig. 4B). A similar phenotype has been detected previously in cells expressing a SCAMP2 mutant (SC2-W202A) or an ADP ribosylation factor 6 (Arf6) mutant (Arf6-N48R), which is deficient in phospholipase D1 (PLD1) activation (24). It has been demonstrated that activation of Arf6 and PLD1 in response to calcium stimulation is important for the synthesis of phosphatidic acid and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]; the latter lipid is an essential factor for priming of DCVs (6, 19, 37, 38). The effect of SCAMP-2 knockdown may reflect abrogation of its association with Arf6 and PLD1 on the plasma membrane (24). Hence, the latency of membrane fusion may be an index for the efficiency of PI(4,5)P2 synthesis at exocytotic sites during priming. In general, the effects of SCAMPs on upstream steps leading to fusion pore opening are likely to be explained by interactions with other proteins. Furthermore, given that interactions with other proteins are selective among SCAMP isoforms (24), it seems likely that individual SCAMPs serve as scaffolds for different proteins during priming.

Role of SCAMP1 and -2 in Fusion Pore Formation Fundamental mechanisms of fusion pore formation are poorly understood, and the study of NE release by amperometry provides important information about pore opening and ensuing dynamic events. Previous observations in both chromaffin and PC12 cells have shown that the nascent narrow fusion pores undergo "flickering" before irreversibly expanding or reclosing. The flickering is reflected by amperometric foot signals, and transient opening and closing registers as a SAF, including large kiss-and-run events (39–41, 43). In our study, we diagnosed SAF (large kiss-and-run events; Fig. 3B) and showed that they are enriched in recordings from cells depleted of SCAMP-1 and -2 alone and in combination (Fig. 5A). Our data analyzed with a simplified three-state model argue that SCAMP-1 and -2 are intimately involved in regulating the fusion step. Knockdown of SCAMP-1 or -2 decreases the rate of dilation (kd) and accelerates the rate of reclosure (kc), with SCAMP-2 knockdown having a stronger relative effect on kc and SCAMP-1 knockdown having a stronger relative effect on kd. Paired SCAMP-1 + SCAMP-2 knockdown decreased kd more significantly, leading to a small extension in open time of the newly created pore (Fig. 5). When these effects are considered along with the observed lack of additive effect on the SAF-to-spike ratio when SCAMP-1 + SCAMP-2 were knocked down together, they suggest that SCAMPs may have sequential action during the development of fusion pores. Accordingly, each of these SCAMPs might have common effects on the membrane but act in series through scaffolding of distinct proteins. Evidently, more experimentation is needed to address such a possibility.

Experimental manipulations such as the expression of synaptotagmins and their mutants (40, 41), elevation of calcium levels (39), and depletion of phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) (13) affect fusion pore {tau} and/or the proportion of fusion pores that open without dilation. Thus, binding of calcium to its sensor and the regulation of PI(4,5)P2 during exocytosis may be key factors that influence the final fusion events. A recent study (7) has shown that the E peptide segments of SCAMP-1 and -2 are able to sequester PI(4,5)P2 via an electrostatic interaction. Furthermore, SCAMP point mutants that are thought to be deficient in PI(4,5)P2 sequestration affect fusion pore duration (21). Hence, interactions of SCAMP-1 and -2 with PI(4,5)P2 and potentially with synaptotagmin, which also binds PI(4,5)P2 (2), may regulate kinetics of fusion pore formation.

Possible Roles of SCAMP-1 and -5 in NE Storage The assay of [3H]NE uptake in PC12 cells has shown that knockdowns of SCAMP-1 and -5 significantly inhibit cellular NE accumulation and that the effect is additive when these SCAMPs are knocked down in tandem. Because no profound differences in the quantal size of released charge between control and knockdowns were detected (Figs. 6 and 7F), the effect on NE uptake does not impact NE efflux from single DCVs. This was a potential concern because recent (although possibly conflicting) studies have reported that knockout of CAPS1, a calcium-triggering protein of exocytosis in chromaffin cells, reduced the quantal size measured for released DCVs due to inhibition of NE loading (12, 33). The lack of effect in our case may reflect at least a couple of possibilities. First, the SCAMP knockdowns may cause a decrease in cytosolic NE or in accumulation in compartments other than DCVs that are either refractory to depolarization-driven discharge or have very different release kinetics. Second, SCAMP-1 and -5 might normally facilitate the storage of NE in DCVs by controlling the trafficking of proteins involved in NE uptake. During the course of knockdown, subpopulations of DCVs that differ in ability to accumulate NE may be created through biogenesis or recycling. DCVs lacking NE would be silent in amperometry measurements, potentially contributing to the decreased frequency of exocytosis (Fig. 4A) while having little impact on quantal size. Recent studies (22, 28) have reported that several SCAMPs bind organellar Na+(K+)/H+ exchanger isoform 7 and that SCAMP2 binds and regulates the endocytic trafficking of serotonin and dopamine transporters. Presently, it is not known whether SCAMP-1 or -5 have corresponding interactions with NE transporters or whether knockdown has the same effect as overexpression, which promoted serotonin transporter accumulation intracellularly rather than on the cell surface (27).

Collaboration Between SCAMP Isoforms in Regulating Exocytosis Our data point to multiple SCAMP isoforms affecting exocytosis, a single trafficking step. Since deficiencies in individual isoforms have effects that are similar yet partially distinct and combined deficiency of SCAMP-1 + SCAMP-2 exhibits additive effects in only selected parameters, how might we rationalize what seem to be individual, nonredundant contributions among isoforms? We view SCAMP isoforms as having supporting functions in DCV exocytosis that are distinct from that of the core fusion machinery [i.e., soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAREs) and SNARE-regulating proteins]. Most likely, SCAMPs collectively serve a combination of roles at the plasma membrane, first, as scaffolds to coordinate the activities of essential exocytotic machinery and, second, to focus and/or organize essential lipids at exocytotic sites. We suggest that the isoforms collaborate in the sense that they bind different proteins that promote the process, e.g., intersectin to SCAMP-1 (10) and the Arf6-PLD1-PI4P5K network to SCAMP-2 (24). Perturbation of these interactions may lead to similar or distinct phenotypes depending on whether the different isoforms act at the same step or sequentially in the priming-triggering-fusion sequence. On the other hand, the highly conserved core structure among SCAMP isoforms (transmembrane spans and adjacent amphipathic segments) (16) suggests that they share a mechanism in maintaining the organization of membranes for fusion events such as exocytosis. In this respect, the specific effects of knockdowns of SCAMP-1 and -2 on fusion pore dynamics may reflect their close proximity to fusion sites. Finally, we raise the possibility that similar lack of redundancy yet collaboration in supporting trafficking events may apply elsewhere within cell surface recycling pathways where SCAMPs reside and are thought to function.


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This work was supported by National Institutes of Health grants, initially DE-09655 and subsequently DK-077380.


   ACKNOWLEDGMENTS
 
We thank Payne Chang, Meyer Jackson, and Edwin Chapman (all at the University of Wisconsin) for providing the computer program for amperometry analysis and PC12 cells. We are grateful to Mary Kate Worden for insightful advice on the presentation of amperometry results, Zhijian Lu for providing the data filter program used for the amperometry analysis, Angela Otero for comments on the manuscript, and Ammasi Periasamy (Keck Imaging Center, University of Virginia) for advice and assistance on the calcium imaging experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Castle, Dept. of Cell Biology, Univ. of Virginia, Charlottesville, VA 22908 (e-mail: jdc4r{at}virginia.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supplemental material for this article is available at the American Journal of Physiology-Cell Physiology website. Back


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SCAMP3 Negatively Regulates Epidermal Growth Factor Receptor Degradation and Promotes Receptor Recycling
Mol. Biol. Cell, March 15, 2009; 20(6): 1816 - 1832.
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