Secretory Granules Are Recaptured Largely Intact After Stimulated Exocytosis in Cultured Endocrine Cells
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Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells Justin W. Taraska*, David Perrais†, Mica Ohara-Imaizumi‡, Shinya Nagamatsu‡, and Wolfhard Almers*§ *Vollum Institute, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201; †Physiologie Cellulaire de la Synapse, Centre National de la Recherche Scientifique Unite´Mixte de Recherche 5091, Universite´Bordeaux 2, Bordeaux 33077, France; and ‡Department of Biochemistry, Kyorin University School of Medicine, Tokyo 181-8611, Japan Communicated by Clay M. Armstrong, University of Pennsylvania School of Medicine, Philadelphia, PA, December 10, 2002 (received for review October 22, 2002) Classical cell biology teaches that exocytosis causes the membrane patterns varied (8), or in permeabilized cells while they overex- of exocytic vesicles to disperse into the cell surface and that a cell pressed proteins involved in exo- and endocytosis (9). In par- must later retrieve by molecular sorting whatever membrane ticular, treatments expected to interfere with the endocytic components it wishes to keep inside. We have tested whether this protein dynamin caused amperometric spikes to broaden and view applies to secretory granules in intact PC-12 cells. Three their charge to increase, suggesting that dynamin caused gran- granule proteins were labeled with fluorescent proteins in differ- ules to reseal before they discharged all their catecholamine (10). ent colors, and two-color evanescent-field microscopy was used to The third approach was based on imaging single granules view single granules during and after exocytosis. Whereas neuro- (11–13). Sea urchin eggs retrieved large vesicles after fertiliza- peptide Y was lost from granules in seconds, tissue plasminogen tion triggered exocytosis (11). In lactotropes, exocytosis caused activator (tPA) and the membrane protein phogrin remained at the granule matrices to bind the externally applied dye FM1-43, and granule site for over 1 min, thus providing markers for postexocytic some granules retained the dye after it was washed from the cell granules. When tPA was imaged simultaneously with cyan fluo- (12). In PC-12 cell membrane patches, exocytosis caused some rescent protein (CFP) as a cytosolic marker, the volume occupied by granules to take up and sequester an extracellular marker, the granule appeared as a dark spot where it excluded CFP. The indicating that the interior of the granule had transiently con- spot remained even after tPA reported exocytosis, indicating that nected to the external space (14). In chromaffin cells, horserad- granules failed to flatten into the cell surface. Phogrin was labeled ish peroxidase appeared in organelles indistinguishable from with GFP at its luminal end and used to sense the pH in granules. granules soon after the stimulation. Once again this was inter- When exocytosis caused the acidic granule interior to neutralize, preted as granules transiently connecting their lumen with the GFP–phogrin at first brightened and later dimmed again as the external space (15). interior separated from the extracellular space and reacidified. The above results provide strong evidence for granule recap- Reacidification and dimming could be reversed by application of ture but questions remain. In the first approach, cases of NH Cl. We conclude that most granules reseal in 10 s after ϩ 4 < fusion-pore closure were too rare at normal external [Ca2 ] (6, releasing cargo, and that these empty or partially empty granules 7) to support a significant role of this mechanism under physi- are recaptured otherwise intact. ologic conditions. This could have been the result of observing only spontaneous fusion events. In the second approach, the PC-12 cells ͉ evanescent-field microscopy ͉ endocytosis ͉ kiss and run observed effects on catecholamine release were subtle, and it was not clear when changes in the amount released reflected changes hen vesicles undergo exocytosis, their membrane must be in the degree of emptying rather than in the catecholamine Wretrieved by endocytosis to maintain a constant cell content of the granule. Finally, in some imaging experiments it surface area. Classical work holds that exocytic vesicles flatten was not clear whether the large vesicles retrieved were empty into the cell surface and allow their components to mix with the granules or vesicles that were newly formed through invagination plasma membrane. These membrane components then are re- of the plasma membrane (11). Other imaging studies explored a trieved by molecular sorting, as occurs during clathrin-mediated time scale of 15–30 min and carried little information on how endocytosis. In contrast, for the stimulated exocytosis of secre- long granule cavities remained open except that closure was tory granules it has been proposed that granules release their unlikely to occur on the millisecond time scale implied by the contents through a fusion pore and then reseal again once cargo duration of amperometric spikes (10). has been released (1, 2). Aside from the loss of cargo and some Here we explore the postexocytic fate of granules when intact membrane components, the granule remains intact and may be and unperturbed PC-12 cells were stimulated by membrane reused or degraded. This mechanism may be referred to as potential changes. Our results confirm three predictions of granule cavity recapture, ‘‘cavicapture’’ (1, 2), or granule recap- granule recapture in PC-12 cells: that a granule membrane ture. A related mechanism was proposed decades ago for protein fails to disperse, that granules keep their shape, and that synaptic vesicles referred to as ‘‘kiss-and-run’’ exocytosis (3). they reseal after exocytosis. Three experimental approaches support granule recapture. In the first, exocytosis of single granules was detected as step Methods increases in membrane capacitance, an assay of cell surface area. Constructs and Cells. Neuropeptide Y (NPY)–enhanced GFP Such studies showed that exocytosis can be reversible in mast (EGFP) and NPY–cyan fluorescent protein (CFP) were made by cells (4, 5). When the release of catecholamine from chromaffin excising the ORF of human pro-NPY by restriction digestion cells was simultaneously detected as an amperometric spike, the from the HindIII and EcoRI sites in NPY–GFP (16) and by spike occurred almost precisely while exocytosis increased the subcloning the fragment into the pEGFP-N1 and pECFP-N1 cell surface area in a step (6, 7). Sometimes, however, a normal parent vectors (CLONTECH͞BD Bioscience, Palo Alto, CA). spike was accompanied by only a transient increase in membrane capacitance, indicating that the connection between granule lumen and the outside, or fusion pore, had opened and closed Abbreviations: NPY, neuropeptide Y; EGFP, enhanced GFP; CFP, cyan fluorescent protein; again (6, 7). The second approach explored how the kinetics of tPA, tissue plasminogen activator; DsRed, Discosoma coral red fluorescent protein. amperometric spikes changed in intact cells while stimulus §To whom correspondence should be addressed. E-mail: [email protected]. 2070–2075 ͉ PNAS ͉ February 18, 2003 ͉ vol. 100 ͉ no. 4 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0337526100 Downloaded by guest on September 27, 2021 To fuse rat tissue plasminogen activator (tPA) to the yellow written in MATLAB. Optical magnification was to 119 nm per fluorescent protein, Venus (tPA–Venus), we removed the cod- pixel except in Fig. 3 (67 nm per pixel). ing sequence of EGFP from tPA–EGFP (17) by digestion with EGFP and DsRed were both excited by the 488-nm laser line AgeI and BsrGI and replaced it with the ORF of Venus (18). The as described (22). Images were acquired for 3.3 min at 2 Hz. To predicted molecular mass of tPA–EGFP is 97 kDa, three times excite CFP and Venus, an acousto-optic modulator (Neos, that of processed NPY–EGFP (31 kDa). Phogrin–Discosoma Melbourne, FL) alternated the laser beam between 458 and 514 coral red fluorescent protein (DsRed) was made by excising the nm. The beam then passed through a custom dual-wavelength phogrin ORF from the phogrin-EGFP plasmid (19) and sub- filter passing the 450–464- and 504–520-nm bands. Light was cloning it into the EcoRI͞AgeI site of the DsRed-N1 vector directed into the objective with a custom dichroic mirror that (CLONTECH). The ORF of rat syntaxin 1A was amplified with reflected from 425 to 462 and 507 to 537 nm and transmitted the Expand high-fidelity PCR system (Roche Molecular Bio- from 463 to 506 and 538 to 614 nm. A 520-nm dichroic mirror chemicals) using the forward primer 5Ј-GAAGATCTCGAGG- (Chroma 520DCLP) in the image splitter separated cyan and GAAGCTTGCCACCATGAAGGACCGAACCCAGGAG- yellow fluorescence. Cyan passed through a 25-nm band-pass and reverse primer 5Ј-CCATCGGGGGCATCTTTGGAGG- filter centered at 480 nm, and yellow passed through a 565 GGTACCCGGGATCCGCG. The PCR product was subse- long-pass filter. Because 458- and 514-nm excitation alternated, quently ligated into the HindIII͞KpnI site of pEGFP-N1 vector the resulting stack had to be deinterleaved off line into 458- and (CLONTECH). We obtained tPA–EGFP from B. Scalettar (Lewis 514-nm stacks. For analysis we used only the cyan image of the and Clark College, Portland, OR); Venus from A. Miyawaki 458-nm stack and the yellow image of the 514-nm stack. Images (RIKEN, Saitama, Japan); Phogrin-EGFP from G. Rutter were acquired at 1 image pair(s). Aside from being imaged with (University of Bristol, Bristol, England); and syntaxin 1A from a camera, cells could also be observed by eye through dual- R. Scheller (Stanford University, Palo Alto, CA). emission filters placed into the eyepieces. Each transmitted from PC-12-GR5 cell stocks (provided by R. Nishi, University of 470 to 496 nm and beyond 533 nm. All filters and mirrors were Vermont, Burlington) were maintained in T80 flasks (Nalgen͞ from Chroma Technology (Brattleboro, VT). ͞ Nunc) at 37°C 10% CO2 in DMEM high glucose (Invitrogen) supplemented with 5% new calf serum and 5% horse serum.