Toward the Inner Nanostructure of a Secretory Vesicle José Luis Nieto-González and Rafael Fernández-Chacón* Instituto de Biomedicina de Sevilla (IBiS, HUVR/CSIC/Universidad de Sevilla), Departamento de Fisiología Médica y Biofísica and CIBERNED, ES-41013 Seville, Spain ABSTRACT: The release of chemical mediators is an essential element of cell-to-cell communication. Signaling molecules such as neurotransmitters and hormones are stored in membrane-bound organelles called secretory vesicles. Some of these organelles can store molecules at high concentrations, overcoming the osmotic shock that could burst the organelle. These organelles contain a proteinaceous matrix that traps the molecules and avoids high intravesicular osmotic pressure. The functional nanostructure and internal organization of the matrix is not well understood. A report by Lovrić et al. in this issue of ACS Nano provides insight into the storage of a small moleculedopaminewithin the intraluminal compartments of a secretory vesicle. Lovrić et al. used a powerful combination of high spatial resolution mass spectrometry and transmission electron microscopy in conjunction with amperometric measurements of exocytotic release to delineate the temporal and spatial fate of intravesicular dopamine and its interaction with the matrix. concentration gradient.3 Secretory vesicles contain a proton pump ATPase that maintains the vesicular lumen acidic at pH ∼ 5.5. Neurotransmitter transporters couple the internalization of specific cargo molecules with the exit of protons from the vesicles. For example, vesicular monoamine transporters (VMAT 1, in chromaffin cells from the adrenal medulla, and VMAT 2, in neurons) bind cytosolic catecholamines (such as adrenaline or dopamine) to be transported into vesicles by exchanging 2H+ ions per catecholamine molecule (Figure 1). The high proton gradient (2 pH units) favors transport activity and enables the vesicle to reach, on average, 1 M catecholamine concentration.4 Such a high concentration of catecholamine molecules, if they were in solution, would lead to significant increases in osmotic pressure and cause membrane tension high enough to break the vesicle membrane. In classical studies using electron microscopy in the central nervous system, researchers classified secretory organelles into two general types: large, dense-core vesicles (LDCVs) and clear vesicles.5 Large, dense-core vesicles are easily identified by their larger size and by the presence of electron-dense material surrounded by a translucent halo area, which occupies most of the vesicle lumen (Figure 1). The dense core is formed by a proteinaceous matrix.6 The clear vesicles, which correspond to the synaptic vesicles that typically store glutamate or GABA, do not have a core and appear not to have any proteinaceous matrix. The proteinaceous matrix is present in secretory vesicles from endocrine cells or the immune system, such as the mast cells.7
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n multicellular organisms, life occurs through the precise and regulated crosstalk between billions of cells. The most universal mode of cell-to-cell communication is based on exocytosis, the release of chemical mediators from one cell to another. In the brain, those mediators are neurotransmitters, and in the endocrine system, the mediators are hormones such as insulin or adrenaline. Chemical mediators are synthesized within cells and packed into membrane-bound organelles called secretory vesicles. Synaptic vesicles are specialized secretory vesicles that release neurotransmitters from the nerve terminals at the synapses. The release of chemical mediators occurs with precise temporal regulation when signaling between cells is required. The arrival of a nerve impulse in a neuron or the chemical activation of an endocrine cell leads to a sudden increase in the cytosolic Ca2+ concentration which, through a sophisticated cascade of protein− protein interactions, triggers the fusion of the secretory vesicle membrane with the plasma membrane.1 The reaction between membranes forms the fusion pore, which enables the contents of the vesicles to be released out to the extracellular media. The fusion pore may open only transiently and then close again, or the membranes may blend completely.2 The quantity of molecules released from the vesicle might depend on the length of time that the fusion pore remains open; however, vesicles need to refill repeatedly to secure efficient exocytosis. Many chemical mediators are small molecules such as acetylcholine, glutamate, γ-aminobutyric acid (GABA), dopamine, or adrenaline. They are first synthesized from a cytosolic precursor and then internalized into the secretory vesicles by specialized transporters. Molecular storage is an important process that requires energy consumption to pump molecules into vesicles against a © 2017 American Chemical Society
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Figure 1. The storage and dynamics of dopamine in a LDCV. The proton pump ATPase maintains the vesicular lumen at low pH. The transporter VMAT uses the proton gradient to pump dopamine into the vesicle. By unknown mechanisms, dopamine seems to leak out through the vesicle membrane. Freshly internalized dopamine is transiently stored, likely in free solution, in the halo compartment (left panel). Long-term storage of dopamine (right panel) takes place in the proteinaceous matrix, formed by granins and other unknown molecules, which enables storage of high dopamine concentration without increasing intravesicular osmotic pressure. For further details, see Lovrić et al. in this issue.12
still limited.12 Secondary ion mass spectrometry (SIMS) is a powerful approach that enables sensitive detection and analysis of chemical elements and their isotopes. The technique depends on a high-energy Cs+ beam that scans the surface and removes secondary particles that are identified based on their mass-tocharge ratios. Interestingly, the beam can be finely focused to perform mass spectrometry imaging at nanometric spatial resolution using NanoSIMS, the nanoscale version of SIMS.13 This option opens enormous possibilities to analyze and to map the distribution of molecules within single cells below 50 nm resolution. The specificity of the molecules to be studied can be achieved by loading cells with isotopically labeled versions of the precursors of the molecules of interest. Because Lovrić et al. were interested in mapping the spatial distribution of dopamine within LDCVs, they incubated PC12 cells (a widely used cell line derived from adrenal medulla) with isotopically labeled L-DOPA (13C-L-DOPA), the precursor of dopamine.12 Upon entering the cell, L-DOPA becomes loaded into the vesicles by the vesicular protein VMAT1. Upon L-DOPA loading, the authors preserved the cells with chemical fixation to carry out the correlative analysis of (1) the biological ultrastructure with transmission electron microscopy (TEM) and (2) the subcellular distribution of freshly internalized isotopically labeled L-DOPA using NanoSIMS. Lovrić et al. used this approach to investigate the cell fate of L-DOPA under different conditions using reserpine, a VMAT blocker known to deplete vesicles of dopamine that has been used in the past to treat high blood pressure in humans. They observed that 13 C-L-DOPA-derived dopamine accumulated into LDCVs after a relatively short (1.5 h) incubation time. In contrast, when Lovrić et al. exposed the cells to reserpine following incubation in 13C-LDOPA, the vesicular enrichment of 13C-L-DOPA-derived dopamine decreased dramatically. The maintenance of dopamine content in LDCVs requires the permanent activity of VMAT because, due to unknown mechanisms, dopamine tends to leak out passively from the vesicle to the cytosol.3 Therefore, those observations suggested that freshly loaded dopamine molecules had a high probability of leaking out. Next, Lovrić et al. used amperometry to quantify the amount of dopamine per vesicle.12 Amperometric measurements in single living cells are achieved by approximating, under the microscope, a carbon fiber microelectrode (few microns in diameter) that is polarized to oxidize monoamines, such as dopamine in this case.14 The same electrode collects the electrons
Several types of proteins have been identified as matrix components, including granins such as chromogranins and secretogranins.8 Granins are single long polypeptides (180− 700 amino acids) with high capacities to bind calcium ions, which induces granin aggregation at low pH. In addition, posttranslational modification of granins, such as sulfations and glycosylation, add negative charge. Granins are pro-hormones that yield smaller peptides with important endocrine functions through proteolysis. The role of granins in the storage of catecholamines in chromaffin cells has been demonstrated in knockout mice lacking chromogranins. The LDCVs in these mice, however, did not completely lose the capacity to store catecholamines, suggesting that, besides chromogranins, other proteins or other intravesicular molecules such as ATP or Ca2+ could be part of the chemical machinery required to maintain isotonicity and the capacity to concentrate catecholamines.9,10 The LDCV matrix has been studied in other cell types as well. The release of serotonin through a transient opening of a fusion pore (i.e., kiss and run), demonstrated in single giant vesicles in mast cells from beige mutant mice, indicates that serotonin was distributed in two compartments within the vesicle.2 One compartment contained serotonin freely in solution, to be released during the transient opening of the fusion pore, whereas another compartment contained the majority of serotonin molecules trapped within the vesicle matrix.2 The release of serotonin has also been measured in isolated secretory vesicles from beige mouse mast cells by inducing the rupture of the vesicle membrane by electroporation.7 Those experiments demonstrated that the efflux of serotonin correlated with the rate of swelling of the granule matrix when the perforated granule was bathed in Ringer’s medium. The results suggested that the vesicle matrix traps and releases serotonin by an ion-exchange mechanism. At low vesicular pH, serotonin is protonated and likely electrostatically attracted by the matrix. The influx of cations will mediate the exchange, with serotonin facilitating its diffusion within the matrix and its release to the extracellular medium.7 In addition, it has been demonstrated that in chromaffin cells, the influx of Na+ through the fusion pore mediates the release of catecholamines.11 Although our understanding of the functional properties of the vesicular matrix has grown over the years, our knowledge about the spatial relationship between the transmitter molecules and the matrix as well as the kinetics governing the movement of molecules within the intravesicular compartments is 3430
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That observation is consistent with the notion that freshly internalized dopamine initially moves to the halo region. The data the authors showed would precisely correspond to the residual fresh dopamine still inside the vesicle after reserpine’s action. It would be useful to see similar analyses in cells incubated under the same conditions but without reserpine. In contrast, vesicles from cells that were incubated in 13C-L-DOPA for 12 h displayed dopamine molecules that were confined to the dense core matrix, indicating that after several hours, the majority of recently internalized molecules became trapped in the matrix core. Those observations support the notion that different regions within the inner structure of the vesicle store dopamine differently, namely, a peripheral halo contains recently acquired dopamine molecules, which are either promptly released or, instead, travel through the halo to the matrix core to join a pool of permanent dopamine molecules (Figure 1). Permanent residency for dopamine molecules is a kinetically slow but reversible process because those molecules will eventually be released by exocytosis. Nevertheless, before exocytosis, permanent resident molecules will be restrained from diffusing freely out of the matrix and will not be able to leak out through the vesicle membrane to the cytosol. This containment is an important mechanism to secure the dopamine cargo without risking the osmotic equilibrium of the vesicle. By using SIMS and TEM simultaneously, Lovrić et al. obtained unprecedented images of dopamine distribution in a plane within a single vesicle with lateral resolution of 50 nm. The work by Lovrić et al. opens interesting possibilities to investigate the nanostructure of vesicles further using SIMS and TEM. The spatial correlations of nanostructures described in the paper bear an unexploited potential for future quantitative studies in different cell conditions. A recent theoretical study proposed a model in which the matrix does not have a homogeneous structure but is instead formed by smaller compacted nanograins occupying the whole vesicle.16 In this model, the nanograins would be more condensed at the matrix core and would take a rather loose conformation within the halo. Perhaps future developments of the nanoSIMS/TEM approach used by Lovrić et al. will help to test that model. An open question in the field is whether synaptic vesicles, along with LDCVs, also contain a proteinaceous matrix to store neurotransmitters. Synaptic vesicles do not appear to have a matrix because they are clear and translucent when observed with electron microscopy. Synaptic vesicles are autonomously recycled at the presynaptic terminals and do not specifically store proteins that require synthesis and processing at the endoplasmic reticulum and the Golgi apparatus, such as granins.17 In addition, proteomic studies in synaptic vesicles that were isolated from the brain did not find any evidence to support the existence of such a polymeric matrix.18 On the other hand, functional and structural evidence for the existence of a matrix has been shown in different types of synaptic vesicles.19 Synaptic vesicles contain transmembrane proteins with polypeptide chains that protrude into the lumen and could hypothetically form a matrix-like structure. Along this idea, the protein SV2, a highly glycosylated synaptic vesicle protein with 12 transmembrane segments, has been proposed as a candidate to form such a proteinaceous matrix.19,20 With such differing evidence, the luminal molecular organization of synaptic vesicles remains unknown. It will be interesting to see if the approaches used by Lovrić et al. develop further to elucidate not only the inner nanostructure of LDCVs but also the inner nanostructure of synaptic vesicles.
In this issue of ACS Nano, Lovric ́ et al. investigated how dopamine is stored within the granule matrix, combining high-resolution spectrometry methods with functional measurements of dopamine release. released during dopamine oxidation, two electrons per molecule, and the integration of the current over time (charge) yields the number of molecules detected. Lovrić et al. found that the amount of dopamine per vesicle increased more than 2-fold in cells incubated in L-DOPA in comparison with cells in basal conditions that were not incubated in L-DOPA. Interestingly, in cells incubated in L-DOPA but treated afterward with reserpine, the amount of vesicular dopamine coincided with the amount existing in basal conditions. Again, that observation supported the notion that the dopamine molecules that were recently internalized are the ones to leak out first. The authors reinforced their conclusions by obtaining similar data based on direct measurements of the vesicular dopamine content in single LDCVs within the cytoplasm using an approach that they previously pioneered, termed intracellular electrochemical cytometry.15 This modality of amperometry uses a nanometersize carbon fiber that penetrates the plasma membrane to measure the dopamine content after individually breaking single vesicles. The authors demonstrated that the vesicles can quickly upload a significant amount of dopamine if the precursor L-DOPA is available; however, such an excess of dopamine is swiftly depleted by reserpine action. Overall, these observations could be explained by considering the existence of two compartments: a soluble compartment that accommodates newly internalized dopamine, and a second compartment, the vesicle core, that functions as a long-term dopamine store. In order to go deeper into that model, Lovrić et al. repeated the experiments with longer incubation times in dopamine: 12 h instead of 1.5 h.12 Interestingly, longer incubations made vesicles more resistant to reserpine-mediated emptying. The authors interpreted this finding as showing that during long incubations, newly internalized dopamine moved from the soluble compartment, the halo, into the core. Such a slow transfer of molecules into the core efficiently prevented the passive leakage of dopamine out of the vesicle. The passive leakage of vesicles through the vesicle membrane is a rather enigmatic and poorly understood phenomenon. Perhaps deprotonated dopamine molecules, which have no net electrical charge, leak out through the phospholipid bilayer more easily than normally protonated molecules. However, it is difficult to deprotonate dopamine within the acidic intravesicular milieu. The advantage of using SIMS and TEM simultaneously is that they combine both sets of data to obtain a high-resolution spatial map that could potentially differentiate the compartments where dopamine accumulates. The authors examined vesicles from two groups of cells that were first incubated in 13 C-L-DOPA, either for 1.5 or 12 h, and then treated with reserpine. In both versions of the experiment, the distribution of 13 C-L-DOPA-derived dopamine was confined to limited areas instead of being spread through the whole vesicular lumen. Interestingly, in vesicles from cells that were incubated for 1.5 h, the distribution of dopamine molecules did not coincide with the dense core matrix that was visualized as the area with the highest electron density in the TEM micrograph. 3431
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(16) Oleinick, A.; Hu, R.; Ren, B.; Tian, Z.-Q.; Svir, I.; Amatore, C. Theoretical Model of Neurotransmitter Release during in Vivo Vesicular Exocytosis Based on a Grainy Biphasic Nano-Structuration of Chromogranins within Dense Core Matrixes. J. Electrochem. Soc. 2016, 163, H3014−H3024. (17) Südhof, T. C. The Synaptic Vesicle Cycle. Annu. Rev. Neurosci. 2004, 27, 509−547. (18) Takamori, S.; Holt, M.; Stenius, K.; Lemke, E. A.; Grønborg, M.; Riedel, D.; Urlaub, H.; Schenck, S.; Brügger, B.; Ringler, P.; Müller, S. A.; Rammber, B.; Gräter, F.; Hub, J. S.; De Groot, B. L.; Mieskes, G.; Moriyama, Y.; Klingauf, J.; Grubmüller, H.; Heuser, J.; et al. Molecular Anatomy of a Trafficking Organelle. Cell 2006, 127, 831−846. (19) Reigada, D.; Díez-Pérez, I.; Gorostiza, P.; Verdaguer, A.; Gómez de Aranda, I.; Pineada, O.; Vilarrasa, J.; Marsal, J.; Blasi, J.; Aleu, J.; Solsona, C. Control of Neurotransmitter Release by an Internal Gel Matrix in Synaptic Vesicles. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 3485−3490. (20) Budzinski, K. L.; Allen, R. W.; Fujimoto, B. S.; Kensel-Hammes, P.; Belnap, D. M.; Bajjalieh, S. M.; Chiu, D. T. Large Structural Change in Isolated Synaptic Vesicles upon Loading with Neurotransmitter. Biophys. J. 2009, 97, 2577−2584.
AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. ORCID
Rafael Fernández-Chacón: 0000-0002-9845-9885 Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS Work in the RFC lab is supported by grants from MINEICO (BFU2013-47493, BFU2016-76050-P), Junta de Andalucía (P12-CTS-2232), ISCIII, and FEDER. REFERENCES (1) Südhof, T. C. The Molecular Machinery of Neurotransmitter Release (Nobel Lecture). Angew. Chem., Int. Ed. 2014, 53, 12696− 12717. (2) Alvarez de Toledo, G.; Fernandez-Chacon, R.; Fernandez, J. M. Release of Secretory Products During Transient Vesicle Fusion. Nature 1993, 363, 554−558. (3) Edwards, R. H. The Neurotransmitter Cycle and Quantal Size. Neuron 2007, 55, 835−58. (4) Gong, L. W.; Hafez, I.; Alvarez de Toledo, G.; Lindau, M. Secretory Vesicles Membrane Area Is Regulated in Tandem with Quantal Size in Chromaffin Cells. J. Neurosci. 2003, 23, 7917−7921. (5) de Robertis, E.; Pellegrino de Iraldi, A. Plurivesicular Secretory Processes and Nerve Endings in the Pineal Gland of the Rat. J. Cell Biol. 1961, 10, 361−372. (6) O’Connor, D. T.; Klein, R. L.; Thureson-Klein, A. K.; Barbosa, J. A.; Chromogranin, A. Localization and Stoichiometry in Large Dense Core Catecholamine Storage Vesicles from Sympathetic Nerve. Brain Res. 1991, 567, 188−196. (7) Marszalek, P. E.; Farrell, B.; Verdugo, P.; Fernandez, J. M. Kinetics of Release of Serotonin from Isolated Secretory Granules. II. Ion Exchange Determines the Diffusivity of Serotonin. Biophys. J. 1997, 73, 1169−1183. (8) Taupenot, L.; Harper, K. L.; O’Connor, D. T. The Chromogranin-Secretogranin Family. N. Engl. J. Med. 2003, 348, 1134−1149. (9) Díaz-Vera, J.; Camacho, M.; Machado, J. D.; Domínguez, N.; Montesinos, M. S.; Hernández-Fernaud, J. R.; Luján, R.; Borges, R.; Chromogranins, A. and B are Key Proteins in Amine Accumulation, but the Catecholamine Secretory Pathway Is Conserved Without Them. FASEB J. 2012, 26, 430−438. (10) Estevez-Herrera, J.; Pardo, M. R.; Dominguez, N.; Pereda, D.; Machado, J. D.; Borges, R. The Role of Chromogranins in the Secretory Pathway. Biomol. Concepts 2013, 4, 605−609. (11) Gong, L. W.; de Toledo, G. A.; Lindau, M. Exocytotic Catecholamine Release Is Not Associated with Cation Flux through Channels in the Vesicle Membrane but Na+ Influx through the Fusion Pore. Nat. Cell Biol. 2007, 9, 915−922. (12) Lovrić, J.; Dunevall, J.; Larsson, A.; Ren, L.; Andersson, S.; Meibom, A.; Malmberg, P.; Kurczy, M. E.; Ewing, A. G. Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles. ACS Nano 2017, DOI: 10.1021/ acsnano.6b07233. (13) Jiang, H.; Kilburn, M. R.; Decelle, J.; Musat, N. NanoSIMS Chemical Imaging Combined with Correlative Microscopy for Biological Sample Analysis. Curr. Opin. Biotechnol. 2016, 41, 130−135. (14) Wightman, R. M.; Jankowski, J. A.; Kennedy, R. T.; Kawagoe, K. T.; Schroeder, T. J.; Leszczyszyn, D. J.; Near, J. A.; Diliberto, E. J., Jr.; Viveros, O. H. Temporally Resolved Catecholamine Spikes Correspond to Single Vesicle Release from Individual Chromaffin Cells. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 10754−10758. (15) Li, X.; Dunevall, J.; Ewing, A. G. Quantitative Chemical Measurements of Vesicular Transmitters with Electrochemical Cytometry. Acc. Chem. Res. 2016, 49, 2347−2354. 3432
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