Clathrin-mediated endocytosis in snake motor terminals is directly facilitated by intracellular Ca2+
1 Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO, USA
Abstract
At the snake neuromuscular junction, low temperature (LT, 5–7°C) blocks clathrin-mediated endocytosis (CME) while exocytosis is largely unaffected. Thus compensatory endocytosis that normally follows transmitter release is inhibited, or ‘delayed’ until the preparation is warmed to room temperature (RT). This delay was exploited to observe how changes in bulk [Ca2+]i directly affect CME. Motor terminals were loaded with fura-2 to monitor [Ca2+]i. With brief stimulation at LT, [Ca2+]i transiently increased but returned to baseline (63 nM) in < 8 min. After 15 min at LT, [Ca2+]i was altered by incubating preparations in the Ca2+ ionophore ionomyocin. Preparations were then warmed to RT to initiate delayed endocytosis, which was quantified as uptake of the fluorescent optical probe sulforhodamine 101. Endocytosis was more rapid when [Ca2+]i increased; the rate at 300 nM Ca2+ was double that under basal conditions. Thus the rate of CME – isolated from stimulation, transmitter release, and other forms of endocytosis – is directly influenced by intraterminal Ca2+.
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Introduction
While it is evident that exocytosis and compensatory endocytosis are matched over time, the means by which endocytosis is regulated to realize this balance remains unclear. One problem in studying such regulation is that release and recycling ordinarily occur simultaneously. Consequently, it is difficult to determine if a factor affects endocytosis directly, or indirectly via some association with release. An example is intraterminal Ca2+. Entry of Ca2+ during stimulation seems to facilitate recycling directly (i.e. independent of the amount of Ca2+-dependent release; Ales et al. 1999; Beutner et al. 2001; Neves et al. 2001; Sankaranarayanan & Ryan, 2001; reviewed by Wu, 2004), consistent with the rapid endocytosis seen during high levels of release at hippocampal synapses (Fernandez-Alfonso & Ryan, 2004). Putative mechanisms underlying this facilitation include the interaction of Ca2+ with synaptotagmin (Poskanzer et al. 2003; Schwarz, 2004) and other Ca2+-sensitive molecules (Chen et al. 2003; Schoch et al. 2004; Smillie & Cousin, 2005). These observations are limited, however, because they relate enhanced endocytosis to conditions in which very high (tens of micromolar; Neher, 1998) levels of Ca2+ exist transiently in microdomains, or hot spots, near active calcium channels at AZs (but see also Cousin & Robinson, 2000). Less is known about the influence of bulk intraterminal Ca2+, which rises to 1 μM or less during activity (250 nM during a 5 Hz tetanus in snake; Teng & Wilkinson, 2003). Bulk levels of Ca2+ are relevant to the substantial endocytosis that occurs after stimulation (when bulk [Ca2+]i remains elevated but hot spots have dissipated), plus all endocytosis that occurs away from the AZ. Yet, in experiments where [Ca2+]i was elevated to 1 μM or less, results have been unclear, with endocytosis sometimes slowed (von Gersdorff & Matthews, 1994) or not measurably affected (Wu & Betz, 1996; Sun et al. 2002; reviewed by Wu, 2004).
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Here we make use of a recent technique, delayed endocytosis, to examine the direct effect of bulk [Ca2+]i on clathrin-mediated endocytosis (CME) at the snake neuromuscular junction. Our technique depends on the snake NMJ's ability to sustain transmitter release at low temperatures (LT, 5–7°C), even though CME is inhibited by blockade of clathrin decoating (Teng & Wilkinson, 2000). Thus, with the preparation cooled, endocytic ‘debt’ builds up with stimulation. The debt, which can remain for hours, is rapidly relieved when endocytosis resumes upon a return to room temperature (RT, 24°C). By altering [Ca2+]i during a period of LT following stimulation, preparations that have been stimulated under identical conditions are returned to RT and undergo CME at prescribed levels of [Ca2+]i. We find that the rate of endocytosis increases substantially with increasing [Ca2+]i. Part of this work has been published in abstract form (Teng & Wilkinson, 2002).
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Methods
Garter snakes (Thamnophis sirtalis) were anaesthetized with pentobarbitol sodium (80 mg kg–1) and killed by rapid decapitation. The single-fibre-thick transversus abdominis muscle comprises > 100 segmental components, each traversing from a rib to the ventral midline and supplied by an individual muscle nerve. Several contiguous segments of the muscle were dissected from the animal, placed in reptilian saline solution and divided as needed to provide two to eight individual nerve–muscle preparations. Each preparation contained three segmental muscles. Details of the muscle's anatomy and of the composition of reptilian saline are given elsewhere (Wilkinson & Lichtman, 1985). All procedures followed the Washington University Guidelines for Animal Studies.
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Electrical stimulation and activity-dependent staining
Nerve–muscle preparations were mounted in a temperature-controlled chamber on the stage of an inverted microscope. The cut end of the muscle nerve was placed in a suction electrode for stimulation (see Teng & Wilkinson, 2003 for details). The central muscle in each preparation was stimulated, with the two adjacent muscles serving as unstimulated controls. Sulforhodamine 101 (SR101, 160 μg ml–1; Lichtman et al. 1985; Teng et al. 1999) was the endocytic probe. Unlike FM1-43, this dye rinses easily from fixed or partially fixed plasma membranes of snake. It was therefore possible to terminate each dye uptake period at a precise time by rapid exchange of the SR101-containing solution with fixative (1% formaldehyde). SR101-containing bath solutions were also exchanged with SR101-free solutions at various times during experiments (exchange time, < 1 s). The brightness of SR101 staining is temperature independent and corresponds to the amount of compensatory endocytosis undergone by a terminal (Teng et al. 1999).
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Measurement of [Ca2+]i
With the nerve–muscle preparation pinned in a dish containing reptilian saline, fura-2 (Molecular Probes, 50 mM) was added to a small well (0.6 μl) formed from silicone sealant. The freshly cut end of the muscle nerve was placed into the well, which was covered with petroleum jelly to prevent drying. The preparation was incubated for 4 h (RT), rinsed with saline and refrigerated (5°C) overnight to permit diffusion and equilibration of the indicator into the nerve terminals (details in Teng & Wilkinson, 2003; see also Wu & Betz, 1996). Ratiometric imaging (340 nm and 380 nm excitation wavelengths) was performed using an inverted microscope equipped with an Intelligent Imaging Innovations (Denver, CO, USA) digital camera system and software. Image pairs were obtained at intervals of 4–6 s. Ratio images were displayed on a computer monitor and regions of individual boutons were manually outlined. The fura-2 dissociation constant at a particular temperature was taken as that given by Larsson et al. (1999). Background fluorescence was averaged from three regions of the muscle fibre surrounding the terminals and subtracted. The corrected image pair ratios were then converted to [Ca2+]i using equations given by Grynkiewicz et al. (1985).
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Control of [Ca2+]i
To change [Ca2+]i the Ca2+ ionophore ionomycin (Sigma Chemical) was applied to the bath. With 1.8 mM extracellular Ca2+, the ion entered boutons along its concentration gradient; [Ca2+]i increased slowly over a 3–4 min incubation period. The rate of increase depended upon ionomycin concentration (2.68–5.36 μM) and bath temperature (LT or RT; see Results).
Measurement of endocytic rate
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Fluorescence of endocytosed SR101 was measured in fixed, slide-mounted preparations on the stage of a Zeiss LSM-510 confocal microscope. Images comprised projections of 9–35 optical sections at 400 nm z-step. Individual boutons were outlined with ‘region of interest’ software (Scion Image for Windows, Scioncorp.com) and the average intensity of pixels within the bouton noted. Boutons from four to eight twitch fibre terminals (58 boutons per terminal) were assayed in each preparation. Background staining of the endplate regions between boutons was subtracted from the average measurement of bouton staining intensity at that NMJ. Total endocytosis was taken as the mean SR101 staining intensity (expressed as 8-bit arbitrary pixel brightness units, ABUs; see Teng & Wilkinson, 2003).
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Results
Two types of experiments are described. Briefly, in calibration experiments, nerve terminals were loaded with fura-2 prior to bath application of the Ca2+ ionophore ionomycin. The dependence of [Ca2+]i upon ionomycin concentration, time of exposure to the drug and temperature was determined by ratiometric imaging. In separate endocytic rate experiments, nerve terminals were stimulated at LT to create endocytic debt. After 15 min, a time sufficient for the Ca2+ transient to subside (Teng & Wilkinson, 2003), the preparation was warmed to RT and the rate of delayed endocytosis was quantified as the amount of SR101 taken up from the bath over a fixed time. Ionomycin was added just before warming to alter [Ca2+]i in a predictable manner, based on calibration experiments.
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Calibration of ionomycin-induced [Ca2+]i increase using fura-2
Because of the known dependence of endocytosis upon levels of extracellular Ca2+ (Teng & Wilkinson, 2003), we altered [Ca2+]i using the method shown in Fig. 1. Ionomycin allowed flux of extracellular Ca2+ into terminals but [Ca2+]o was not changed, nor was sufficient time allowed for [Ca2+]i to equilibrate with [Ca2+]o. Rather, [Ca2+]o was held at physiological level (1.8 mM) throughout the experiments and, consequently, [Ca2+]i increased continuously after addition of ionomycin. The ionomyocin concentrations chosen (2.68 μM and 5.36 μM) provided ideal rates of intraterminal Ca2+ increase without muscle contraction, which would have interfered with imaging.
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A, pseudocolour image of two motor terminals filled with fura-2 (scale at right). At LT (above) [Ca2+]i was 20 nM in terminals and axons. After bath application of 5.36 μM ionomycin (centre), [Ca2+]i rose in terminals but not axons. After a temperature step to RT (below), [Ca2+]i rose to 60 nM in terminal 2 and 90 nM in terminal 1. B, plot of monotonic rise in [Ca2+]i with time after ionomycin. Note difference between adjacent terminals 1 and 2, rapid increase with shift from LT to RT, and relative stability of [Ca2+]i at RT. C, averaged results from all experiments using either 2.68 μM (circles) or 5.36 μM ionomycin (triangles) measured from images before (0 s) and after application of ionomycin. Baseline [Ca2+]i is normalized to its average (62.5 nM; see text). Error bars are S.D.
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One to three adjacent terminals were imaged simultaneously. An example is in Fig. 1A. In the three pseudocolour micrographs, taken at the times indicated, brightness represents fura-2 emission at 380 nm excitation while hue represents the Ca2+ concentration calculated from the 340 nm/380 nm excitation ratio. The muscle nerve branch, containing several axons, is at the right. In the upper panel (LT; before addition of ionomycin to the bath) the nerve branch and two axonal terminals all contain roughly the same baseline [Ca2+]i of 20 nm. After 50 s, ionomycin (5.36 μM) was applied. The middle panel shows the consequent slow Ca2+ increase at 3 min after application of the drug. A second, quicker rise in [Ca2+]i occurred immediately after elevation of bath temperature to RT, particularly in terminal 1 (lower panel). Figure 1B shows the entire sequence of Ca2+ measurements, taken at 3–4 s intervals, from the two terminals of Fig. 1A. [Ca2+]i rose slowly with time, particularly after the jump at the transition to RT. Thus, over a brief (40 s) period at RT a quasi-stable Ca2+ level could be defined. Interestingly, that level varied among terminals, even among those supplied by the same muscle nerve. In this example [Ca2+]i tripled in terminal 2, but quadrupled in terminal 1. These characteristics, including variability, were seen in all terminals in three snakes examined with ionomycin concentrations of 2.68 μM (n = 7 terminals) and 5.36 μM (n = 4 terminals). Figure 1C summarizes the calibration results (all using the protocol of Fig. 1B). Measured baseline [Ca2+]i varied substantially among terminals (62.5 ± 5.7 nM; mean ± S.E.M.; n = 99 terminals). After ionomycin, the calcium level increased slowly and monotonically at LT in all terminals, then underwent a quick further increase when the bath temperature was stepped to RT. We noticed that terminals with higher measured baseline [Ca2+]i had higher [Ca2+]i after ionomycin, and vice versa. Thus the factor by which calcium level increased was somewhat less scattered among terminals than the measured levels (both baseline and after ionomycin) themselves. The average [Ca2+]i during the 40 s RT period was 105 nM ± 22 nM (mean ± S.D.) for 2.68 μM ionomycin concentration and 303 nM ± 110 nM for 5.36 μM ionomycin.
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Measurement of endocytic rate
Endocytosis proceeds very slowly in snake terminals stimulated and held at LT, and remains incomplete for hours unless temperature is increased to RT. Once at RT, rapid ‘delayed’ endocytosis begins immediately. For the stimulus protocol (300 stimuli; 5 Hz for 60 s) and delay time (15 min) used in the present study, delayed endocytosis requires 90 s to complete at normal (1.8 mM) bath [Ca2+] (Teng & Wilkinson, 2003). We therefore measured endocytic rate by quantifying SR101 uptake during a 40 s temperature pulse to RT. By allowing insufficient time for endocytosis to be completed, any increase or decrease in endocytic rate due to experimental manipulation appeared as an increase or decrease in SR101 staining intensity.
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After stimulation and 18 min delay at LT, the bath was exchanged for one containing SR101 at RT. Exactly 3 min prior to the exchange, ionomycin was added (0 μM control, 2.68 μM or 5.36 μM). The RT bath remained for 40 s, at which time the preparation was washed in cold fixative (same timing as the protocol used in Ca2+ calibration measurements of Fig. 1). Results from one experiment are shown in Fig. 2. Each micrograph (Fig. 2A–C) shows a small part of one terminal (3–5 of 58 boutons) in one of 12 muscles, all from the same snake. Four muscles were untreated with ionomycin (0 μM; Fig. 2A), four received 2.68 μM ionomycin (Fig. 2B), and four received 5.36 μM ionomycin (Fig. 2C). The left panels (RT stim) show typical SR101 uptake in terminals during the RT period. The rate of endocytosis increased markedly with increasing ionomycin concentration. Adjacent unstimulated muscles in the same preparations (RT unstim) lacked endocytic debt and exhibited far less dye uptake. Moreover, this ‘background’ dye uptake lacked significant dependence on ionomycin concentration. As stated above, ionomycin was added 3 min at LT prior to the temperature pulse to RT. As expected, very slow endocytosis occurred at LT in stimulated preparations, and we found that this rate was also sensitive to ionomycin. To observe this, SR101 was added to the bath along with ionomycin for 3 min at LT; the preparation was then fixed without warming (Fig. 2, LT stim). Staining intensity increased slightly compared to unstimulated controls (Fig. 2, LT unstim). Note that, while all of the images of Fig. 2A–C were obtained under identical conditions, SR101 incubation times at LT were increased to 3 min (versus 40 s for those at RT) in order to provide a better dye signal. Thus, for a given endocytic rate, the brightness of the LT images in Fig. 2 is enhanced 4.5-fold as compared to those at RT.
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One experiment compared 12 nerve–muscle preparations from one snake. A–C, each micrograph shows a few typical boutons from one preparation. A, control SR101 uptake without ionomycin (0 μM). Robust delayed endocytosis after the step to RT produced punctate staining in stimulated (RT stim) but not unstimulated (RT unstim) muscles. Boutons in stimulated muscles kept at LT endocytosed weakly (LT stim). Boutons in unstimulated muscles that were kept at LT showed extremely weak background staining (LT unstim). B, addition of ionomycin (2.68 μM) after stimulation increased delayed endocytic rate (RT stim) substantially. Endocytosis at LT also increased (LT stim). Background endocytosis (RT unstim, LT unstim) was little changed. C, increased ionomycin concentration (5.36 μM) further increased endocytic rate in stimulated boutons (RT stim; LT stim) but had little effect on background endocytosis (RT unstim; LT unstim). Brightness of LT preparations is enhanced 4.5-fold by longer dye incubation times (see text). Shown below A–C is the protocol timing for RT (left) and LT (right) measurements. D–F, results from all boutons imaged. Each plot corresponds to the micrographs above it; each data point is the mean SR101 uptake from 68 to 141 boutons in one muscle (ABU; error bars are S.D.). D, SR101 uptake at RT (delayed endocytosis) increased substantially with increasing ionomycin concentration. E, background staining in unstimulated RT preparations increased only slightly with ionomycin. F, endocytosis at LT in stimulated terminals also increased with ionomycin. G, background endocytosis at LT, like that at RT, increased sligntly. Staining brightness in stimulated preparations (D and F) is plotted with background staining (E and G, respectively) subtracted.
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Results from all boutons imaged in the experiment of Fig. 2 (68–141 per muscle) are presented in Fig. 2D–G. At RT, ionomycin at either concentration increased SR101 background staining in unstimulated controls slightly (P < 0.01; Fig. 2E), perhaps because of increased miniature endplate potential activity (and consequent compensatory endocytosis) with elevated [Ca2+]i. In contrast, uptake of SR101 increased substantially with increasing ionomycin concentration in the stimulated muscle (P < 0.01). The averaged results are in Fig. 2D. Background staining at each ionomycin concentration (Fig. 2E; described above) has been subtracted; thus the dependence of dye uptake on ionomycin shown in Fig. 2D is due exclusively to delayed endocytosis. As stated above, there was little dye uptake at LT. Background staining (unstimulated) is shown in Fig. 2G; ionomycin had no substantial effect. In contrast, ionomycin at either concentration significantly increased staining (LT endocytic rate) in stimulated boutons (P < 0.01; Fig. 2F; background is subtracted as in Fig. 2D). The difference in staining between the two ionomycin concentrations was significant at RT but not LT. As in the images of Fig. 2A–C, SR101 incubation times were 3 min at LT (Fig. 2F and G) and 40 s at RT (Fig. 2D and E).
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The averaged results from three experiments using the protocol of Fig. 2 are presented in Fig. 3. Unstimulated (background) staining data are not shown, but have been subtracted as in Fig. 2. Data are also normalized for the difference in incubation times at RT and LT, and are presented as endocytic rates (ABU s–1). The rate of delayed endocytosis (RT) approximately doubled when [Ca2+]i increased from 60 nM to 300 nM. A similar dependence of endocytosis upon [Ca2+]i was seen at LT (Fig. 3, circles), even though endocytosis was slowed by more than an order of magnitude compared to that at RT. Each data point in Fig. 3 was averaged from 282 to 406 boutons. Dependence of uptake on ionomycin concentration (and thus on [Ca2+]i; see below) was significant at both RT (P < 0.01) and LT (P < 0.01).
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[Ca2+]i levels on the abcissa are from calibration experiments. The ordinate of each data point is averaged from 282 to 406 boutons in 3 snakes and is corrected for background staining as in Fig. 2. Rate of SR101 endocytosis is reported as average increase in pixel brightness (arbitrary units, ABU) for each second of incubation. RT endocytosis increased with increasing [Ca2+]i (P < 0.01); LT endocytosis increased significantly (P < 0.01) only at higher [Ca2+]i. Error bars are S.D.
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Ionomycin has no effect on endocytosis in a Ca2+ free bath
We confirmed that ionomycin's effect was due exclusively to intracellular Ca2+ increase by repeating the experiments of Figs 1 and 2 in a bath containing no added Ca2+ and 5 mM EGTA. Results are presented in Fig. 4. Fura-2 imaging of terminals showed no significant change from baseline [Ca2+]i during incubation with 5.36 μM ionomycin at both LT and RT (example in Fig. 4A; compare to Fig. 1B). Averaged results from measurements during the RT period at 0, 5.36 and 20.1 μM ionomycin are in Fig. 4B (compare to data at 300 s in Fig. 1C). Addition of ionomycin to a Ca2+ free bath had little or no effect upon [Ca2+]i over the time scale of our experiments (n = 4 terminals in 1 muscle at 5.36 μM ionomycin, P > 0.14; n = 4 terminals in 1 muscle at 20.1 μM ionomycin, P < 0.02; see Discussion).
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A, typical experiment using protocol of Fig. 1B. [Ca2+]i did not change from baseline when 5.36 μM ionomycin was applied at LT, nor when the preparation was warmed to RT (compare to Fig. 1B). B, [Ca2+]i averaged from 4 terminals during the RT period of the protocol of Fig. 1B. [Ca2+]i did not substantially change from baseline, even when ionomycin concentration was elevated to 20.1 μM.C, rate of SR101 uptake at RT was lower than in normal bath [Ca2+]o due to dependence of endocytosis upon extracellular Ca2+, but was not altered significantly by ionomycin (compare to Fig. 3).
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Similarly, in endocytic rate experiments using the protocol of Fig. 2, we saw no significant change in endocytosis at LT (data not shown) or at RT when 5.36 μM ionomycin was added to a Ca2+ free bath (Fig. 4C; n = 358 boutons, 3 muscles; P > 0.05). Thus the effect of ionomycin upon endocytic rate required extracellular Ca2+ and therefore depended upon the increase in [Ca2+]i provided by the ionophore. Note that, although the endocytic rate at 0 mM Ca2+ (0.6 ABU s–1; Fig. 4C) was not influenced by ionomycin, it was 20% less than the comparable endocytic rate at the same baseline [Ca2+]i in a bath containing 1.8 mM Ca2+ (0.75 ABU s–1; Fig. 3). This difference was presumably due to the direct influence of [Ca2+]o upon endocytic rate (Teng & Wilkinson, 2003).
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Discussion
Three principal types of recycling occur in nerve terminals – CME, macropinocytosis (with subsequent vesicle budding via CME) and ‘kiss-and-run’. In addition, the terms ‘fast’ and ‘slow’ are used, based on kinetic measurements. Macropinocytosis is probably the slowest type (de Lange et al. 2003). Fast recycling is associated with kiss-and-run (e.g. Ales et al. 1999) although the relationships among rate-limiting kinetics and physiological processes (e.g. CME) are not known. Kinetic regulation via Ca2+ could facilitate a given type (e.g. CME), or, alternatively, switch endocytosis to a faster type (Ales et al. 1999; Beutner et al. 2001; Neves et al. 2001; de Lange et al. 2003). However, especially when kinetic but not optical measurements are made, it is difficult to determine what types of endocytosis are involved.
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Our method avoids these ambiguities. Delayed endocytosis is CME because it is initiated by release from the low temperature blockade of clathrin decoating (Teng & Wilkinson, 2000). The punctate SR101 staining pattern associated with delayed endocytosis (Fig. 2A) is consistent with CME (Teng & Wilkinson, 2000). There is no evidence of a transition to kiss-and-run, which would decrease, not increase, dye uptake (e.g. Fernandez-Alfonso & Ryan, 2004). Because Ca2+ enters slowly and continuously over minutes, there are no Ca2+ hot spots. Moreover, similar to the Drosophilia shibire NMJ after return to permissive temperature (reviewed by Kidokoro et al. 2004), we have observed no difference in time course or staining pattern between delayed endocytosis and CME that occurs without delay upon stimulation at RT. Thus we conclude that the most ubiquitous form of endocytosis, CME, is directly facilitated by the physiologically elevated levels of Ca2+ that exist throughout the terminal during neural activity.
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We found previously that extracellular Ca2+ influences endocytosis; in those experiments [Ca2+]i was constant at baseline levels (Teng & Wilkinson, 2003). Conversely, in the present work [Ca2+]o was constant so that only [Ca2+]i could potentially influence endocytic rate. The sensitivity of endocytosis to change in [Ca2+]i was robust, but less than the fourth-power dependence exhibited by exocytosis (Dodge & Rahamimoff, 1967). At RT, a 4-fold increase in [Ca2+]i from baseline resulted in a 3-fold increase in endocytic rate, approximately a linear relationship (slope of Fig. 3 RT data on double logarithmic plot, 0.68). Although endocytosis was extremely slow at LT, it was similarly influenced by [Ca2+]i (double logarithmic slope, 0.52). Thus the dependence of CME on Ca2+ remained even when CME was slowed by clathrin decoating blockade.
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We also attempted to decrease [Ca2+]i below baseline by adding ionomycin to a 0 mM Ca2+, 5 mM EGTA bath. There was no effect over 30 min (i.e. a longer time than that of the control experiments of Fig. 4), presumably due to intracellular buffering by mitochondria (David et al. 1998). Consequently, we do not know if endocytic rate can be reduced below that seen at baseline [Ca2+]i.
Bulk intracellular Ca2+ is one of several mechanisms, like intracellular Cl– (Hull & von Gersdorff, 2004), extracellular Ca2+ (Ales et al. 1999; Teng & Wilkinson, 2003) or protein phosphatases (Cousin & Robinson, 2001), that potentially coregulate exocytosis, endocytosis and plasma membrane area in nerve terminals. Delayed endocytosis might serve as a useful tool for study of these mechanisms, and others as they are identified.
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Abstract
At the snake neuromuscular junction, low temperature (LT, 5–7°C) blocks clathrin-mediated endocytosis (CME) while exocytosis is largely unaffected. Thus compensatory endocytosis that normally follows transmitter release is inhibited, or ‘delayed’ until the preparation is warmed to room temperature (RT). This delay was exploited to observe how changes in bulk [Ca2+]i directly affect CME. Motor terminals were loaded with fura-2 to monitor [Ca2+]i. With brief stimulation at LT, [Ca2+]i transiently increased but returned to baseline (63 nM) in < 8 min. After 15 min at LT, [Ca2+]i was altered by incubating preparations in the Ca2+ ionophore ionomyocin. Preparations were then warmed to RT to initiate delayed endocytosis, which was quantified as uptake of the fluorescent optical probe sulforhodamine 101. Endocytosis was more rapid when [Ca2+]i increased; the rate at 300 nM Ca2+ was double that under basal conditions. Thus the rate of CME – isolated from stimulation, transmitter release, and other forms of endocytosis – is directly influenced by intraterminal Ca2+.
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Introduction
While it is evident that exocytosis and compensatory endocytosis are matched over time, the means by which endocytosis is regulated to realize this balance remains unclear. One problem in studying such regulation is that release and recycling ordinarily occur simultaneously. Consequently, it is difficult to determine if a factor affects endocytosis directly, or indirectly via some association with release. An example is intraterminal Ca2+. Entry of Ca2+ during stimulation seems to facilitate recycling directly (i.e. independent of the amount of Ca2+-dependent release; Ales et al. 1999; Beutner et al. 2001; Neves et al. 2001; Sankaranarayanan & Ryan, 2001; reviewed by Wu, 2004), consistent with the rapid endocytosis seen during high levels of release at hippocampal synapses (Fernandez-Alfonso & Ryan, 2004). Putative mechanisms underlying this facilitation include the interaction of Ca2+ with synaptotagmin (Poskanzer et al. 2003; Schwarz, 2004) and other Ca2+-sensitive molecules (Chen et al. 2003; Schoch et al. 2004; Smillie & Cousin, 2005). These observations are limited, however, because they relate enhanced endocytosis to conditions in which very high (tens of micromolar; Neher, 1998) levels of Ca2+ exist transiently in microdomains, or hot spots, near active calcium channels at AZs (but see also Cousin & Robinson, 2000). Less is known about the influence of bulk intraterminal Ca2+, which rises to 1 μM or less during activity (250 nM during a 5 Hz tetanus in snake; Teng & Wilkinson, 2003). Bulk levels of Ca2+ are relevant to the substantial endocytosis that occurs after stimulation (when bulk [Ca2+]i remains elevated but hot spots have dissipated), plus all endocytosis that occurs away from the AZ. Yet, in experiments where [Ca2+]i was elevated to 1 μM or less, results have been unclear, with endocytosis sometimes slowed (von Gersdorff & Matthews, 1994) or not measurably affected (Wu & Betz, 1996; Sun et al. 2002; reviewed by Wu, 2004).
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Here we make use of a recent technique, delayed endocytosis, to examine the direct effect of bulk [Ca2+]i on clathrin-mediated endocytosis (CME) at the snake neuromuscular junction. Our technique depends on the snake NMJ's ability to sustain transmitter release at low temperatures (LT, 5–7°C), even though CME is inhibited by blockade of clathrin decoating (Teng & Wilkinson, 2000). Thus, with the preparation cooled, endocytic ‘debt’ builds up with stimulation. The debt, which can remain for hours, is rapidly relieved when endocytosis resumes upon a return to room temperature (RT, 24°C). By altering [Ca2+]i during a period of LT following stimulation, preparations that have been stimulated under identical conditions are returned to RT and undergo CME at prescribed levels of [Ca2+]i. We find that the rate of endocytosis increases substantially with increasing [Ca2+]i. Part of this work has been published in abstract form (Teng & Wilkinson, 2002).
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Methods
Garter snakes (Thamnophis sirtalis) were anaesthetized with pentobarbitol sodium (80 mg kg–1) and killed by rapid decapitation. The single-fibre-thick transversus abdominis muscle comprises > 100 segmental components, each traversing from a rib to the ventral midline and supplied by an individual muscle nerve. Several contiguous segments of the muscle were dissected from the animal, placed in reptilian saline solution and divided as needed to provide two to eight individual nerve–muscle preparations. Each preparation contained three segmental muscles. Details of the muscle's anatomy and of the composition of reptilian saline are given elsewhere (Wilkinson & Lichtman, 1985). All procedures followed the Washington University Guidelines for Animal Studies.
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Electrical stimulation and activity-dependent staining
Nerve–muscle preparations were mounted in a temperature-controlled chamber on the stage of an inverted microscope. The cut end of the muscle nerve was placed in a suction electrode for stimulation (see Teng & Wilkinson, 2003 for details). The central muscle in each preparation was stimulated, with the two adjacent muscles serving as unstimulated controls. Sulforhodamine 101 (SR101, 160 μg ml–1; Lichtman et al. 1985; Teng et al. 1999) was the endocytic probe. Unlike FM1-43, this dye rinses easily from fixed or partially fixed plasma membranes of snake. It was therefore possible to terminate each dye uptake period at a precise time by rapid exchange of the SR101-containing solution with fixative (1% formaldehyde). SR101-containing bath solutions were also exchanged with SR101-free solutions at various times during experiments (exchange time, < 1 s). The brightness of SR101 staining is temperature independent and corresponds to the amount of compensatory endocytosis undergone by a terminal (Teng et al. 1999).
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Measurement of [Ca2+]i
With the nerve–muscle preparation pinned in a dish containing reptilian saline, fura-2 (Molecular Probes, 50 mM) was added to a small well (0.6 μl) formed from silicone sealant. The freshly cut end of the muscle nerve was placed into the well, which was covered with petroleum jelly to prevent drying. The preparation was incubated for 4 h (RT), rinsed with saline and refrigerated (5°C) overnight to permit diffusion and equilibration of the indicator into the nerve terminals (details in Teng & Wilkinson, 2003; see also Wu & Betz, 1996). Ratiometric imaging (340 nm and 380 nm excitation wavelengths) was performed using an inverted microscope equipped with an Intelligent Imaging Innovations (Denver, CO, USA) digital camera system and software. Image pairs were obtained at intervals of 4–6 s. Ratio images were displayed on a computer monitor and regions of individual boutons were manually outlined. The fura-2 dissociation constant at a particular temperature was taken as that given by Larsson et al. (1999). Background fluorescence was averaged from three regions of the muscle fibre surrounding the terminals and subtracted. The corrected image pair ratios were then converted to [Ca2+]i using equations given by Grynkiewicz et al. (1985).
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Control of [Ca2+]i
To change [Ca2+]i the Ca2+ ionophore ionomycin (Sigma Chemical) was applied to the bath. With 1.8 mM extracellular Ca2+, the ion entered boutons along its concentration gradient; [Ca2+]i increased slowly over a 3–4 min incubation period. The rate of increase depended upon ionomycin concentration (2.68–5.36 μM) and bath temperature (LT or RT; see Results).
Measurement of endocytic rate
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Fluorescence of endocytosed SR101 was measured in fixed, slide-mounted preparations on the stage of a Zeiss LSM-510 confocal microscope. Images comprised projections of 9–35 optical sections at 400 nm z-step. Individual boutons were outlined with ‘region of interest’ software (Scion Image for Windows, Scioncorp.com) and the average intensity of pixels within the bouton noted. Boutons from four to eight twitch fibre terminals (58 boutons per terminal) were assayed in each preparation. Background staining of the endplate regions between boutons was subtracted from the average measurement of bouton staining intensity at that NMJ. Total endocytosis was taken as the mean SR101 staining intensity (expressed as 8-bit arbitrary pixel brightness units, ABUs; see Teng & Wilkinson, 2003).
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Results
Two types of experiments are described. Briefly, in calibration experiments, nerve terminals were loaded with fura-2 prior to bath application of the Ca2+ ionophore ionomycin. The dependence of [Ca2+]i upon ionomycin concentration, time of exposure to the drug and temperature was determined by ratiometric imaging. In separate endocytic rate experiments, nerve terminals were stimulated at LT to create endocytic debt. After 15 min, a time sufficient for the Ca2+ transient to subside (Teng & Wilkinson, 2003), the preparation was warmed to RT and the rate of delayed endocytosis was quantified as the amount of SR101 taken up from the bath over a fixed time. Ionomycin was added just before warming to alter [Ca2+]i in a predictable manner, based on calibration experiments.
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Calibration of ionomycin-induced [Ca2+]i increase using fura-2
Because of the known dependence of endocytosis upon levels of extracellular Ca2+ (Teng & Wilkinson, 2003), we altered [Ca2+]i using the method shown in Fig. 1. Ionomycin allowed flux of extracellular Ca2+ into terminals but [Ca2+]o was not changed, nor was sufficient time allowed for [Ca2+]i to equilibrate with [Ca2+]o. Rather, [Ca2+]o was held at physiological level (1.8 mM) throughout the experiments and, consequently, [Ca2+]i increased continuously after addition of ionomycin. The ionomyocin concentrations chosen (2.68 μM and 5.36 μM) provided ideal rates of intraterminal Ca2+ increase without muscle contraction, which would have interfered with imaging.
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A, pseudocolour image of two motor terminals filled with fura-2 (scale at right). At LT (above) [Ca2+]i was 20 nM in terminals and axons. After bath application of 5.36 μM ionomycin (centre), [Ca2+]i rose in terminals but not axons. After a temperature step to RT (below), [Ca2+]i rose to 60 nM in terminal 2 and 90 nM in terminal 1. B, plot of monotonic rise in [Ca2+]i with time after ionomycin. Note difference between adjacent terminals 1 and 2, rapid increase with shift from LT to RT, and relative stability of [Ca2+]i at RT. C, averaged results from all experiments using either 2.68 μM (circles) or 5.36 μM ionomycin (triangles) measured from images before (0 s) and after application of ionomycin. Baseline [Ca2+]i is normalized to its average (62.5 nM; see text). Error bars are S.D.
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One to three adjacent terminals were imaged simultaneously. An example is in Fig. 1A. In the three pseudocolour micrographs, taken at the times indicated, brightness represents fura-2 emission at 380 nm excitation while hue represents the Ca2+ concentration calculated from the 340 nm/380 nm excitation ratio. The muscle nerve branch, containing several axons, is at the right. In the upper panel (LT; before addition of ionomycin to the bath) the nerve branch and two axonal terminals all contain roughly the same baseline [Ca2+]i of 20 nm. After 50 s, ionomycin (5.36 μM) was applied. The middle panel shows the consequent slow Ca2+ increase at 3 min after application of the drug. A second, quicker rise in [Ca2+]i occurred immediately after elevation of bath temperature to RT, particularly in terminal 1 (lower panel). Figure 1B shows the entire sequence of Ca2+ measurements, taken at 3–4 s intervals, from the two terminals of Fig. 1A. [Ca2+]i rose slowly with time, particularly after the jump at the transition to RT. Thus, over a brief (40 s) period at RT a quasi-stable Ca2+ level could be defined. Interestingly, that level varied among terminals, even among those supplied by the same muscle nerve. In this example [Ca2+]i tripled in terminal 2, but quadrupled in terminal 1. These characteristics, including variability, were seen in all terminals in three snakes examined with ionomycin concentrations of 2.68 μM (n = 7 terminals) and 5.36 μM (n = 4 terminals). Figure 1C summarizes the calibration results (all using the protocol of Fig. 1B). Measured baseline [Ca2+]i varied substantially among terminals (62.5 ± 5.7 nM; mean ± S.E.M.; n = 99 terminals). After ionomycin, the calcium level increased slowly and monotonically at LT in all terminals, then underwent a quick further increase when the bath temperature was stepped to RT. We noticed that terminals with higher measured baseline [Ca2+]i had higher [Ca2+]i after ionomycin, and vice versa. Thus the factor by which calcium level increased was somewhat less scattered among terminals than the measured levels (both baseline and after ionomycin) themselves. The average [Ca2+]i during the 40 s RT period was 105 nM ± 22 nM (mean ± S.D.) for 2.68 μM ionomycin concentration and 303 nM ± 110 nM for 5.36 μM ionomycin.
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Measurement of endocytic rate
Endocytosis proceeds very slowly in snake terminals stimulated and held at LT, and remains incomplete for hours unless temperature is increased to RT. Once at RT, rapid ‘delayed’ endocytosis begins immediately. For the stimulus protocol (300 stimuli; 5 Hz for 60 s) and delay time (15 min) used in the present study, delayed endocytosis requires 90 s to complete at normal (1.8 mM) bath [Ca2+] (Teng & Wilkinson, 2003). We therefore measured endocytic rate by quantifying SR101 uptake during a 40 s temperature pulse to RT. By allowing insufficient time for endocytosis to be completed, any increase or decrease in endocytic rate due to experimental manipulation appeared as an increase or decrease in SR101 staining intensity.
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After stimulation and 18 min delay at LT, the bath was exchanged for one containing SR101 at RT. Exactly 3 min prior to the exchange, ionomycin was added (0 μM control, 2.68 μM or 5.36 μM). The RT bath remained for 40 s, at which time the preparation was washed in cold fixative (same timing as the protocol used in Ca2+ calibration measurements of Fig. 1). Results from one experiment are shown in Fig. 2. Each micrograph (Fig. 2A–C) shows a small part of one terminal (3–5 of 58 boutons) in one of 12 muscles, all from the same snake. Four muscles were untreated with ionomycin (0 μM; Fig. 2A), four received 2.68 μM ionomycin (Fig. 2B), and four received 5.36 μM ionomycin (Fig. 2C). The left panels (RT stim) show typical SR101 uptake in terminals during the RT period. The rate of endocytosis increased markedly with increasing ionomycin concentration. Adjacent unstimulated muscles in the same preparations (RT unstim) lacked endocytic debt and exhibited far less dye uptake. Moreover, this ‘background’ dye uptake lacked significant dependence on ionomycin concentration. As stated above, ionomycin was added 3 min at LT prior to the temperature pulse to RT. As expected, very slow endocytosis occurred at LT in stimulated preparations, and we found that this rate was also sensitive to ionomycin. To observe this, SR101 was added to the bath along with ionomycin for 3 min at LT; the preparation was then fixed without warming (Fig. 2, LT stim). Staining intensity increased slightly compared to unstimulated controls (Fig. 2, LT unstim). Note that, while all of the images of Fig. 2A–C were obtained under identical conditions, SR101 incubation times at LT were increased to 3 min (versus 40 s for those at RT) in order to provide a better dye signal. Thus, for a given endocytic rate, the brightness of the LT images in Fig. 2 is enhanced 4.5-fold as compared to those at RT.
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One experiment compared 12 nerve–muscle preparations from one snake. A–C, each micrograph shows a few typical boutons from one preparation. A, control SR101 uptake without ionomycin (0 μM). Robust delayed endocytosis after the step to RT produced punctate staining in stimulated (RT stim) but not unstimulated (RT unstim) muscles. Boutons in stimulated muscles kept at LT endocytosed weakly (LT stim). Boutons in unstimulated muscles that were kept at LT showed extremely weak background staining (LT unstim). B, addition of ionomycin (2.68 μM) after stimulation increased delayed endocytic rate (RT stim) substantially. Endocytosis at LT also increased (LT stim). Background endocytosis (RT unstim, LT unstim) was little changed. C, increased ionomycin concentration (5.36 μM) further increased endocytic rate in stimulated boutons (RT stim; LT stim) but had little effect on background endocytosis (RT unstim; LT unstim). Brightness of LT preparations is enhanced 4.5-fold by longer dye incubation times (see text). Shown below A–C is the protocol timing for RT (left) and LT (right) measurements. D–F, results from all boutons imaged. Each plot corresponds to the micrographs above it; each data point is the mean SR101 uptake from 68 to 141 boutons in one muscle (ABU; error bars are S.D.). D, SR101 uptake at RT (delayed endocytosis) increased substantially with increasing ionomycin concentration. E, background staining in unstimulated RT preparations increased only slightly with ionomycin. F, endocytosis at LT in stimulated terminals also increased with ionomycin. G, background endocytosis at LT, like that at RT, increased sligntly. Staining brightness in stimulated preparations (D and F) is plotted with background staining (E and G, respectively) subtracted.
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Results from all boutons imaged in the experiment of Fig. 2 (68–141 per muscle) are presented in Fig. 2D–G. At RT, ionomycin at either concentration increased SR101 background staining in unstimulated controls slightly (P < 0.01; Fig. 2E), perhaps because of increased miniature endplate potential activity (and consequent compensatory endocytosis) with elevated [Ca2+]i. In contrast, uptake of SR101 increased substantially with increasing ionomycin concentration in the stimulated muscle (P < 0.01). The averaged results are in Fig. 2D. Background staining at each ionomycin concentration (Fig. 2E; described above) has been subtracted; thus the dependence of dye uptake on ionomycin shown in Fig. 2D is due exclusively to delayed endocytosis. As stated above, there was little dye uptake at LT. Background staining (unstimulated) is shown in Fig. 2G; ionomycin had no substantial effect. In contrast, ionomycin at either concentration significantly increased staining (LT endocytic rate) in stimulated boutons (P < 0.01; Fig. 2F; background is subtracted as in Fig. 2D). The difference in staining between the two ionomycin concentrations was significant at RT but not LT. As in the images of Fig. 2A–C, SR101 incubation times were 3 min at LT (Fig. 2F and G) and 40 s at RT (Fig. 2D and E).
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The averaged results from three experiments using the protocol of Fig. 2 are presented in Fig. 3. Unstimulated (background) staining data are not shown, but have been subtracted as in Fig. 2. Data are also normalized for the difference in incubation times at RT and LT, and are presented as endocytic rates (ABU s–1). The rate of delayed endocytosis (RT) approximately doubled when [Ca2+]i increased from 60 nM to 300 nM. A similar dependence of endocytosis upon [Ca2+]i was seen at LT (Fig. 3, circles), even though endocytosis was slowed by more than an order of magnitude compared to that at RT. Each data point in Fig. 3 was averaged from 282 to 406 boutons. Dependence of uptake on ionomycin concentration (and thus on [Ca2+]i; see below) was significant at both RT (P < 0.01) and LT (P < 0.01).
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[Ca2+]i levels on the abcissa are from calibration experiments. The ordinate of each data point is averaged from 282 to 406 boutons in 3 snakes and is corrected for background staining as in Fig. 2. Rate of SR101 endocytosis is reported as average increase in pixel brightness (arbitrary units, ABU) for each second of incubation. RT endocytosis increased with increasing [Ca2+]i (P < 0.01); LT endocytosis increased significantly (P < 0.01) only at higher [Ca2+]i. Error bars are S.D.
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Ionomycin has no effect on endocytosis in a Ca2+ free bath
We confirmed that ionomycin's effect was due exclusively to intracellular Ca2+ increase by repeating the experiments of Figs 1 and 2 in a bath containing no added Ca2+ and 5 mM EGTA. Results are presented in Fig. 4. Fura-2 imaging of terminals showed no significant change from baseline [Ca2+]i during incubation with 5.36 μM ionomycin at both LT and RT (example in Fig. 4A; compare to Fig. 1B). Averaged results from measurements during the RT period at 0, 5.36 and 20.1 μM ionomycin are in Fig. 4B (compare to data at 300 s in Fig. 1C). Addition of ionomycin to a Ca2+ free bath had little or no effect upon [Ca2+]i over the time scale of our experiments (n = 4 terminals in 1 muscle at 5.36 μM ionomycin, P > 0.14; n = 4 terminals in 1 muscle at 20.1 μM ionomycin, P < 0.02; see Discussion).
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A, typical experiment using protocol of Fig. 1B. [Ca2+]i did not change from baseline when 5.36 μM ionomycin was applied at LT, nor when the preparation was warmed to RT (compare to Fig. 1B). B, [Ca2+]i averaged from 4 terminals during the RT period of the protocol of Fig. 1B. [Ca2+]i did not substantially change from baseline, even when ionomycin concentration was elevated to 20.1 μM.C, rate of SR101 uptake at RT was lower than in normal bath [Ca2+]o due to dependence of endocytosis upon extracellular Ca2+, but was not altered significantly by ionomycin (compare to Fig. 3).
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Similarly, in endocytic rate experiments using the protocol of Fig. 2, we saw no significant change in endocytosis at LT (data not shown) or at RT when 5.36 μM ionomycin was added to a Ca2+ free bath (Fig. 4C; n = 358 boutons, 3 muscles; P > 0.05). Thus the effect of ionomycin upon endocytic rate required extracellular Ca2+ and therefore depended upon the increase in [Ca2+]i provided by the ionophore. Note that, although the endocytic rate at 0 mM Ca2+ (0.6 ABU s–1; Fig. 4C) was not influenced by ionomycin, it was 20% less than the comparable endocytic rate at the same baseline [Ca2+]i in a bath containing 1.8 mM Ca2+ (0.75 ABU s–1; Fig. 3). This difference was presumably due to the direct influence of [Ca2+]o upon endocytic rate (Teng & Wilkinson, 2003).
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Discussion
Three principal types of recycling occur in nerve terminals – CME, macropinocytosis (with subsequent vesicle budding via CME) and ‘kiss-and-run’. In addition, the terms ‘fast’ and ‘slow’ are used, based on kinetic measurements. Macropinocytosis is probably the slowest type (de Lange et al. 2003). Fast recycling is associated with kiss-and-run (e.g. Ales et al. 1999) although the relationships among rate-limiting kinetics and physiological processes (e.g. CME) are not known. Kinetic regulation via Ca2+ could facilitate a given type (e.g. CME), or, alternatively, switch endocytosis to a faster type (Ales et al. 1999; Beutner et al. 2001; Neves et al. 2001; de Lange et al. 2003). However, especially when kinetic but not optical measurements are made, it is difficult to determine what types of endocytosis are involved.
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Our method avoids these ambiguities. Delayed endocytosis is CME because it is initiated by release from the low temperature blockade of clathrin decoating (Teng & Wilkinson, 2000). The punctate SR101 staining pattern associated with delayed endocytosis (Fig. 2A) is consistent with CME (Teng & Wilkinson, 2000). There is no evidence of a transition to kiss-and-run, which would decrease, not increase, dye uptake (e.g. Fernandez-Alfonso & Ryan, 2004). Because Ca2+ enters slowly and continuously over minutes, there are no Ca2+ hot spots. Moreover, similar to the Drosophilia shibire NMJ after return to permissive temperature (reviewed by Kidokoro et al. 2004), we have observed no difference in time course or staining pattern between delayed endocytosis and CME that occurs without delay upon stimulation at RT. Thus we conclude that the most ubiquitous form of endocytosis, CME, is directly facilitated by the physiologically elevated levels of Ca2+ that exist throughout the terminal during neural activity.
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We found previously that extracellular Ca2+ influences endocytosis; in those experiments [Ca2+]i was constant at baseline levels (Teng & Wilkinson, 2003). Conversely, in the present work [Ca2+]o was constant so that only [Ca2+]i could potentially influence endocytic rate. The sensitivity of endocytosis to change in [Ca2+]i was robust, but less than the fourth-power dependence exhibited by exocytosis (Dodge & Rahamimoff, 1967). At RT, a 4-fold increase in [Ca2+]i from baseline resulted in a 3-fold increase in endocytic rate, approximately a linear relationship (slope of Fig. 3 RT data on double logarithmic plot, 0.68). Although endocytosis was extremely slow at LT, it was similarly influenced by [Ca2+]i (double logarithmic slope, 0.52). Thus the dependence of CME on Ca2+ remained even when CME was slowed by clathrin decoating blockade.
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We also attempted to decrease [Ca2+]i below baseline by adding ionomycin to a 0 mM Ca2+, 5 mM EGTA bath. There was no effect over 30 min (i.e. a longer time than that of the control experiments of Fig. 4), presumably due to intracellular buffering by mitochondria (David et al. 1998). Consequently, we do not know if endocytic rate can be reduced below that seen at baseline [Ca2+]i.
Bulk intracellular Ca2+ is one of several mechanisms, like intracellular Cl– (Hull & von Gersdorff, 2004), extracellular Ca2+ (Ales et al. 1999; Teng & Wilkinson, 2003) or protein phosphatases (Cousin & Robinson, 2001), that potentially coregulate exocytosis, endocytosis and plasma membrane area in nerve terminals. Delayed endocytosis might serve as a useful tool for study of these mechanisms, and others as they are identified.
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