Article |
Address correspondence to Sanford M. Simon, Laboratory of Cellular Biophysics, Rockefeller University, Box 304, 1230 York Ave., New York, NY 10021. Tel.: (212) 327-8130. Fax: (212) 327-7543. E-mail: simon{at}rockefeller.edu
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Abstract |
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Key Words: TIR-FM; CD63; dextran; VAMP7; Synaptotagmin VII
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Introduction |
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Unlike the specialized secretory cells, the cellular and molecular details of calcium-dependent exocytosis in nonsecretory cells are still poorly understood. The lack of consensus regarding the cellular compartment responsible for calcium-regulated exocytosis in nonsecretory cells has been an important hurdle in deciphering further details of this process. On one hand, it has been proposed that specialized synaptic-like vesicles are responsible for the calcium-regulated exocytosis in CHO fibroblasts (Chavez et al., 1996); on the other hand, organelles such as endosomes (Eddleman et al., 1998), Golgi-derived vesicles (Togo et al., 1999), and lysosomes (Rodriguez et al., 1997) have been claimed to be the vesicles responsible for this process.
Calcium influx triggered by membrane damage leads to lysosomal exocytosis in a variety of mammalian cells (Reddy et al., 2001). Exocytosis of lysosomes has also been observed in response to the increase in cytosolic calcium caused by invading pathogens such as Trypanosoma cruzi (Caler et al., 1998, 2000) and Neisseria (Ayala et al., 2001). Nonetheless, it is still unresolved whether only one or various organelles in nonsecretory cells undergo calcium-regulated exocytosis. Furthermore, obstruction of kinesin- and myosin-driven vesicular transport (Bi et al., 1997) or of actin depolymerization (Miyake et al., 2001) negatively affects the calcium-dependent exocytosis, thereby inhibiting membrane resealing in nonsecretory cells. Thus, it also remains to be elucidated if the role of calcium is to trigger exocytosis of vesicles that are docked at the plasma membrane, or if calcium is responsible for the long range movement, tethering or docking of vesicles at the site of fusion. To address these questions it is important to identify and observe, in situ, the behavior of calcium regulated exocytic compartments in nonsecretory cells.
Total internal reflection fluorescence microscopy (TIR-FM)* offers the ability to visualize events occurring near the cell surface with a high signal-to-noise ratio (Axelrod, 1981). Earlier work from our and other laboratories has shown that this is a powerful technique for the study of exocytosis (Schmoranzer et al., 2000). Here we have used TIR-FM to study, in live cells, the behavior of fluorescently labeled Golgi apparatus, Golgi-derived vesicles, ER, early endosomes, late endosomes, and lysosomes in response to calcium stimulation. Lysosomes were the only organelles we observed to undergo calcium- induced exocytosis in a variety of nonsecretory cells. Calcium primarily triggered the exocytosis of a pool of lysosomes that were immediately apposed and appear docked to the plasma membrane.
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Results |
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Using epifluorescence and TIR-FM microscopy, we observed that compartments containing calreticulin-EYFP (Fig. 1 A, diagnostic of ER), human growth hormoneEGFP conjugated to four FM domains (Fig. 1 B, diagnostic of post Golgi derived vesicles), VAMP8-ECFP (Fig. 1 D, early endosomes), Rab7-ECFP (Fig. 1 E, late endosomes), and CD63-EGFP (Fig. 1 F, lysosomes), were present both deeper in the cell and within the evanescent field. In contrast, the galactosyl-transferase-ECFP (Fig. 1 C, diagnostic of Golgi apparatus; Sciaky et al., 1997; Llopis et al., 1998; Zaal et al., 1999)-labeled compartments were observed only deeper inside the cell and were not visible in the evanescent field.
With the exception of the lysosomes and Golgi apparatus, all of the other organelles mentioned above, including the endoplasmic reticulum (Lysakowski et al., 1999; Nagata, 2001), post-Golgi vesicles (Schmoranzer et al., 2000) and endosomes (Lampson et al., 2001) have been observed within 100 nm from the plasma membrane. Because the existence of a population of lysosomes adjacent to the plasma membrane has not been characterized, we tested if additional lysosomal markers were observed in the evanescent field (Fig. 2 B). With each of the fluorescently tagged lysosomal membrane proteins used, synaptotagmin VII, Lamp1, and VAMP7, we observed a population of vesicles adjacent to the plasma membrane, within the evanescent field (Fig. 2 B). Further, to examine if a peripheral population of lysosomes was typical of only CHO cells, we repeated the experiment in various cell lines (Fig. 2 C). All the cell lines examined (NIH3T3 fibroblasts, WM239 melanoma, MDCK epithelial cells, normal rat kidney (NRK) fibroblasts, HeLa cells, and murine embryonic primary fibroblasts) possessed a fraction of dextran labeled lysosomes adjacent to the plasma membrane.
Increase in calcium triggers exocytosis of lysosomes, but not of other organelles
To assess the effect of calcium on the movement and distribution of the fluorescently tagged compartments in the evanescent field, and on their ability to exocytose, cellular calcium was increased using the calcium ionophore A23187 (10 µM; Bennett et al., 1979). After the ionophore treatment, cells were continuously imaged (at 510 frames/s) using TIR-FM. Observations were limited to an interval of <10 min, because treatment with the calcium ionophore A23187 often caused cells to round up after longer periods, leading to their disappearance from the evanescent field. Within this interval, the vesicles were analyzed for movement and fusion based on total fluorescence intensity, peak intensity, and the width squared ([width]2) of the spread of fluorescence. As demonstrated previously (Schmoranzer et al., 2000), movement of a fluorescent vesicle perpendicular to the coverslip alters the excitation by evanescent field, thereby resulting in changes in the fluorescence emission intensity. A fusion event, leading to the delivery of all membrane proteins of a vesicle to the plasma membrane, is determined by two criteria. First, there is an increase in the peak fluorescence intensity and the total fluorescence intensity (as all fluorophores are delivered to the plasma membrane, and hence better excited by the evanescent wave). Second, there is an increase in the width of the spread of fluorescence (as the vesicular membrane proteins diffuses into the plasma membrane; Schmoranzer et al., 2000). Upon fusion the rate at which the (width)2 of the fluorescence increases is linear with time, the slope of which is equal to the diffusion constant for the membrane protein. If a vesicle lysed, the (width)2 would increase significantly faster and there would be no net delivery of fluorophores to the plasma membrane.
The organelles and vesicles we examined after stimulation of the cells with calcium ionophore fell into three groups. In one group, the vesicles stopped moving and became stationary adjacent to the membrane, or were stationary during the entire period of observation. In the second group, the vesicles showed synchronous increases and decreases in the total and peak fluorescent intensities, with no significant change in the width. The third group of vesicles showed synchronous increase in total and peak intensities, and a concomitant increase in the width of fluorescence. These results are consistent with the first group of vesicles docking with the plasma membrane but not fusing during the observation period; the second group of vesicles moving in and out of the plane of the evanescent field, without fusing with the plasma membrane; and the third group fusing to the plasma membrane.
In untreated cells, we did not observe the Golgi apparatus in the evanescent field (n = 4; Fig. 1 C). The endoplasmic reticulum (n = 5), early endosomes (n = 5), late endosomes (n = 6), and lysosomes (n = 23) were present in the evanescent field (Fig. 1, A, D, E, and F). Based on the above mentioned criteria, these compartments fell into the first or the second group of vesicles: no exocytosis was observed. Post-Golgi transport vesicles were also seen in the evanescent field (n = 6) in CHO cells (Fig. 1 B), which exocytosed at the rate of 6 ± 2/min (n = 4). Treatment with calcium ionophore did not affect the rate of fusion of the post-Golgi vesicles. The Golgi apparatus, ER, early endosomes, and late endosomes were never observed to exocytose in the presence or absence of calcium ionophore (Table II). Further, calcium affected neither the movement nor the number of any of these organelles in the evanescent field.
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To identify which of the two populations of lysosomes, perinuclear or plasma membrane apposed, was responsible for exocytosis we tracked individual lysosomes in CHO cells and murine embryonic primary fibroblasts. Both in the CHO cells (Fig. 6 A) and primary fibroblasts (Fig. 6 B), >80% of the lysosomes that exocytosed were present in the evanescent field before the increase in cytosolic calcium. Moreover, whereas in untreated CHO cells 35% of the lysosomes present in the evanescent field were motile, addition of calcium ionophore led to a rapid loss of movement of these lysosomes (n = 21) (Fig. 6 C). Increase in calcium did not lead to a significant increase in the recruitment of lysosomes to the vicinity of the plasma membrane, but instead (due to exocytosis), over a period of 510 min there was a 4 ± 1% decrease in the total number of lysosomes in the evanescent field. Further, the majority of lysosomes in CHO cells (210 out of 270, [81%]) that underwent exocytosis did not move before undergoing fusion. Similar observations were made with the primary fibroblasts. Thus, addition of the calcium ionophore seems to cause the exocytosis of lysosomes that are apparently docked at the plasma membrane.
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Discussion |
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An increase in calcium caused either by the calcium ionophore A23187 or by the IP3-dependent calcium channel agonists thrombin and bombesin, led to exocytosis of lysosomes. Using dual-color live-cell imaging to simultaneously visualize the presence of membrane and lumenal markers, we found that calcium induced the exocytosis of both lysosomal membrane and lumenal markers (Fig. 3 D; Video 2). Identification of lysosomes as the vesicles responsible for A23187-dependent exocytosis is in agreement with the earlier reports of calcium-induced lysosomal exocytosis in fibroblast and epithelial cells (Reddy et al., 2001). It is also in agreement with the size (0.41.5 µm) and density (1.12 g/ml) reported for a calcium-regulated exocytic compartment present in CHO cells (Chavez, et al., 1996; Ninomiya et al., 1996).
Most of the lysosomes that underwent calcium-dependent exocytosis are those that are present near the plasma membrane (Fig 6, A and B; Videos 1 and 3). Moreover, an increase in calcium also led to a reduction in the fraction of motile lysosomes in the evanescent field (Fig. 6 C). Thus, similar to the calcium-regulated exocytic vesicles present in specialized secretory cells, in nonsecretory cells, calcium appears to be primarily responsible for the fusion rather than recruitment of lysosomes to the plasma membrane. Although in CHO and other cell lines only a small proportion of the membrane proximal lysosomes underwent fusion, in primary fibroblasts, this number was significantly higher, reaching 24% (Table III). Because exocytosis of lysosomes has been implicated in the process of membrane repair (Reddy et al., 2001), the cell-typespecific differences in the extent of lysosomal exocytosis observed here may correspond to the cell-typespecific sensitivity to calcium previously reported in membrane resealing assays (Steinhardt et al., 1994). On the other hand, we cannot exclude the possibility that different cell types differ in their sensitivity to the calcium ionophore. It is important to note that, unlike a rupture in the plasma membrane, treatment with calcium ionophores does not equilibrate the cytosolic calcium with the extracellular calcium (Kendall et al., 1996). Therefore, it remains to be determined if calcium influx during a membrane wound, which results in free intracellular calcium concentrations at the millimolar level, triggers the exocytosis of a larger fraction of the lysosomal population.
A good example of cell lineage-specific differences in the extent of lysosomal exocytosis is provided by the secretory lysosomes of hemopoietic cells, which can correspond to as much as 60% of the total lysosomal population. In hemopoietic cells, secretory lysosomes have the unique ability to store specific secretory proteins in their lysosome-like granules, while maintaining its endocytic and degradative capabilities (Stinchcombe and Griffiths, 1999; Blott and Griffiths, 2002). Our results reveal a remarkable similarity between calcium regulated exocytic vesicles from nonsecretory and those present in specialized secretory cells, in so far that both are peripherally located granule population with lysosomal properties and that they respond to calcium by fusing with the plasma membrane (Marks and Seabra, 2001; Blott and Griffiths, 2002).
Calcium-induced exocytosis is important in the repair of membrane rupture and the invasion of cells by some pathogens (Caler et al., 2000; Reddy et al., 2001). In this study, total internal reflection microscopy allowed us to directly visualize a peripheral population of lysosomes (Fig. 2, B and C) that shows calcium-dependent exocytosis (Figs. 3 and 4). This opens the possibility of studying the mechanistic and kinetic details of membrane repair and pathogen invasion. The observation that primary fibroblasts show more extensive calcium induced lysosomal exocytosis than immortalized cultured cells (Fig. 6, A and B; Table III) indicates that a peripheral population of lysosomes that undergoes calcium induced exocytosis is not an aberration of immortalized cell lines in culture. Future studies should clarify if regulated lysosomal exocytosis is more pronounced in primary cells due to its critical role in maintaining plasma membrane integrity under mechanically challenging conditions. In any case, demonstration of a membrane docked pool of lysosomes as the compartment responsible for calcium-induced exocytosis clearly necessitates a revaluation of the physiological functions of this organelle.
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Materials and methods |
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Cell treatments
Murine embryonic primary fibroblasts were prepared from day 13.5 embryos of C57BL/6J mice, as described in Tournier et al. (2000), and cultured in DME supplemented with 10% FBS. All experiments were done with cells between passages 1 and 2. CHO fibroblasts, NRK fibroblasts, HeLa cells, MDCK epithelial, CHO and NRK cells were maintained in DME (Mediatech Cellgro) supplemented with 10% FBS, and WM239 melanoma cells were maintained in RPMI (GIBCO BRL) supplemented with 10% FBS, in a 37°C incubator humidified with 5% CO2. Cells were plated on sterile coverslips (Fisher Scientific) and just before imaging the media was replaced with cell imaging media (CIM; HBSS + 10 mM Hepes + 5% FBS, pH 7.4). Cells were transiently transfected using Lipofectamine 2000 (Invitrogen) or Fugene 6 (Roche) 1836 h before being imaged. For calcium measurements, CHO cells were loaded for 20 min with 10 µM Fluo-3AM and 10 µM Fura redAM, washed three times, and maintained in CIM, after which 10 µM A23187 calcium-ionophore was added (T0s) and the cells were imaged using TIRF microscopy. For calcium ionophore and calcium agonist treatments, growth media was replaced with CIM and mounted on the microscope stage maintained at 37°C. Cells were imaged using TIRFM before and after the addition of 10 µM A23187 ionophore or 0.2 U/ml thrombin or 20 nM bombesin. Calcium ionophore-A23187, thrombin, bombesin and 65 kD TRITC dextran were obtained from Sigma-Aldrich. 10 kD Texas reddextran and 70 kD FITC dextran were obtained from Molecular Probes, and used to load lysosomes as previously described (Martinez et al., 2000).
Image acquisition
Laser scanning confocal microscopy.
Laser scanning confocal microscopy was carried out using a Zeiss LSM 510 confocal microscope using a 63X (Plan-Apochromat, NA 1.40) water immersion objective. GFP was imaged using 488-nm laser light and a 505530-nm BP emission filter, CFP was imaged using 458-nm laser excitation and 460500-nm BP emission filter, Texas red and TRITC were imaged using 543-nm laser excitation and a 580-nm LP emission filter. Serial optical sections were taken using 0.4-µm optical sections.
TIR-FM.
The illumination for TIR-FM was done through the objective as previously described (Schmoranzer et al., 2000). It consists of an inverted epifluorescence microscope (IX-70; Olympus) equipped with high numerical aperture lenses (Apo 60X NA 1.45; Olympus) and a home-built temperature-controlled enclosure. GFP tagged proteins were excited with the 488-nm line of an Argon laser (Omnichrome, model 543-AP A01; Melles Griot) reflected off a dichroic mirror (498DCLP). For simultaneous dual-color imaging of GFP/Texas red imaging or Fluo-3/Fura red, we used an emission splitter (W-view; Hamamatsu Photonics). Cells were excited using the 488-nm line of Argon laser. The GFP emission was collected through emission band pass filters (HQ525/50M). The GFP/TR or the Fluo-3/Fura red emissions were collected simultaneously through an emission splitter equipped with dichroic mirrors to split the emission (550DCLP) and emission band pass filters (GFP/Fluo-3, HQ525/50M; TR/Fura red, HQ580LP). All filters were obtained from Chroma Technologies Corp. Images were acquired with a 12-bit cooled CCD ORCA-ER (Hamamatsu Photonics) with a resolution of 1280 x 1024 pixels (pixel size, 6.45 µm2). The camera and mechanical shutters (Uniblitz, Vincent Associates) were controlled using MetaMorph (Universal Imaging). Images were acquired at 510 frames/s. Images containing a region of interest of the cell were streamed to memory on a PC during acquisition and then saved to hard disk. The depth of the evanescent filed was typically 70120 nm for the Apo 60x N.A. 1.45 lens (Schmoranzer et al., 2000).
Image processing and quantitative analysis.
Processing and analysis of the video sequences was done either with MetaMorph or using in-house software written in LabView. For processing dual-color sequences, the images acquired through the emission splitter such that the separated channels appear side by side on the camera chip were separated, subtracted for background fluorescence, aligned within accuracy of a single pixel using the brightfield or fluorescence images and analyzed using MetaMorph. Finally, the separate channels were pseudo-color encoded and combined in a RGB sequence. Quantitation of fluorescence intensity and (half-[width])2 was done as described earlier (Schmoranzer et al., 2000).
Online supplemental material
All videos are available at http://www.jcb.org/cgi/content/full/jcb.200208154/DC1. Video 1 corresponds with Fig. 2 C and shows that calcium induces exocytosis of lysosomes in murine embryonic primary fibroblasts. Video 2 corresponds with Fig. 3 D and shows that lysosomal exocytosis leads to the release of both membrane and lumenal content. Video 3 corresponds to Fig. 4 B and shows that calcium induces exocytosis of only a small fraction of lysosomes.
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Footnotes |
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* Abbreviations used in this paper: FM, FK506 modified; LAMP, lysomal-associated membrane protein; NRK, normal rat kidney; TIR-FM, total internal reflectionfluorescence microscopy; VAMP, vesicle-associated membrane protein.
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Acknowledgments |
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This work was supported by National Science Foundation grants BES 0110070 and BES-0119468 to S.M. Simon, and National Institutes of Health grant R01 GM64625 TO N.W. Andrews.
Submitted: 26 August 2002
Revised: 22 October 2002
Accepted: 23 October 2002
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