Basal and Stress-Induced Network Activity in the Adrenal Medulla In Vivo.

The adrenal medulla plays a critical role in mammalian homeostasis and the stress response. It is populated by clustered chromaffin cells that secrete epinephrine or norepinephrine along with peptides into the bloodstream affecting distant target organs. Despite been heavily studied, the central control of adrenal medulla and in-situ spatiotemporal responsiveness remains poorly understood. For this work, we continuously monitored the electrical activity of individual adrenomedullary chromaffin cells in the living anesthetized rat using multielectrode arrays. We measured the chromaffin cell activity under basal and physiological stress conditions and characterized the functional micro-architecture of the adrenal medulla. Under basal conditions, chromaffin cells fired action potentials with frequencies between ~0.2 and 4 Hz. Activity was almost completely driven by sympathetic inputs coming through the splanchnic nerve. Chromaffin cells were organized into independent local networks in which cells fired in a specific order, with latencies from hundreds of microseconds to a few milliseconds. Electrical stimulation of the splanchnic nerve evoked almost exactly the same spatiotemporal firing patterns that occurred spontaneously. Hypoglycemic stress, induced by insulin administration resulted in increased activity of a subset of the chromaffin cells. In contrast, respiratory arrest induced by lethal anesthesia resulted in an increase in the activity of virtually all chromaffin cells before cessation of all activity. These results suggest a stressor-specific activation of adrenomedullary chromaffin cell networks and revealed a surprisingly complex electrical organization that likely reflects the dynamic nature of the adrenal medulla's neuroendocrine output during basal conditions and during different types of physiological stress.

VAMP2 and synaptotagmin mobility in chromaffin granule membranes: implications for regulated exocytosis.

Granule-plasma membrane docking and fusion can only occur when proteins that enable these reactions are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may influence docking and membrane fusion. We measured the mobility of vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field produced by total internal reflection was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, enabling the measurement of diffusion coefficients. We found that VAMP2-EGFP and Syt7-EGFP are mobile with a diffusion coefficient of ∼3 × 10-10 cm2/s. Syt1-EGFP mobility was below the detection limit. Utilizing these diffusion parameters, we estimated the time required for these proteins to arrive at docking and nascent fusion sites to be many tens of milliseconds. Our analyses raise the possibility that the diffusion characteristics of VAMP2 and Syt proteins could be a factor that influences the rate of exocytosis.

Roles for the SNAP25 linker domain in the fusion pore and a dynamic plasma membrane SNARE "acceptor" complex.

Central to the exocytotic release of hormones and neurotransmitters is the interaction of four SNARE motifs in proteins on the secretory granule/synaptic vesicle membrane (synaptobrevin/VAMP, v-SNARE) and on the plasma membrane (syntaxin and SNAP25, t-SNAREs). The interaction is thought to bring the opposing membranes together to enable fusion. An underlying motivation for this Viewpoint is to synthesize from recent diverse studies possible new insights about these events. We focus on a recent paper that demonstrates the importance of the linker region joining the two SNARE motifs of the neuronal t-SNARE SNAP25 for maintaining rates of secretion with roles for distinct segments in speeding fusion pore expansion. Remarkably, lipid-perturbing agents rescue a palmitoylation-deficient mutant whose phenotype includes slow fusion pore expansion, suggesting that protein-protein interactions have a role not only in bringing together the granule or vesicle membrane with the plasma membrane but also in orchestrating protein-lipid interactions leading to the fusion reaction. Unexpectedly, biochemical investigations demonstrate the importance of the C-terminal domain of the linker in the formation of the plasma membrane t-SNARE "acceptor" complex for synaptobrevin2. This insight, together with biophysical and optical studies from other laboratories, suggests that the plasma membrane SNARE acceptor complex between SNAP25 and syntaxin and the subsequent trans-SNARE complex with the v-SNARE synaptobrevin form within 100 ms before fusion.

Dynamic Relationship of the SNARE Complex with a Membrane.

Fusion of secretory granules and synaptic vesicles with the plasma membrane is driven by SNARE protein interactions. Intensive investigations in vitro, which include x-ray crystallography, cryoelectron microscopy, and NMR analyses by numerous groups, have elucidated structures relevant to the function of these proteins. Although function depends on the proteins being membrane bound, for experimental reasons, most of the studies have used cytosolic domains, as exemplified by the groundbreaking studies that elucidated the structure of a tetrapeptide helical bundle formed by interaction of the cytosolic domains of syntaxin1A, SNAP25 (two peptides) and synaptobrevin 2. Because the cytosolic fragments were unfettered by membrane attachments, it is likely that the tetrapeptide helical bundle reflects the lowest energy state, such as that found in the "cis" interactions of the SNARE motifs after fusion when they co-localize in the plasma membrane. Much more difficult to study and still poorly understood are critical "trans" interactions between the synaptic vesicle SNARE protein synaptobrevin 2 and the plasma membrane syntaxin1A/SNAP25 complex that initiate the fusion event. In a series of articles from the laboratory of Lukas Tamm, the spontaneous orientation of the SNARE motif of membrane-bound, full-length syntaxin1A with respect to the membrane hosting syntaxin's transmembrane domain was investigated with nanometer precision under a variety of conditions, including those that model aspects of the "trans" configuration. The studies rely on fluorescence interference-contrast microscopy, a technique that utilizes the pattern of constructive and destructive interference arising from incoming and reflected excitation and emission light at the surface of a silicon chip that has been layered with oxidized silicon of varying depths. This Perspective discusses their findings, including the unexpected influence of the degree of lipid unsaturation on the orientation of the SNARE complex.

Identification of ß-synuclein on secretory granules in chromaffin cells and the effects of α- and ß-synuclein on post-fusion BDNF discharge and fusion pore expansion.

α-Synuclein is strongly implicated in the pathogenesis of Parkinson's disease as well as in other neurodegenerative diseases. However, its normal function in cells is not understood. The N-termini of α-, ß-, and γ-synuclein contains six to seven 11-amino acid repeats that are predicted to form amphipathic helices. Membrane-binding and membrane-curving abilities of synuclein raise the possibility that synuclein could alter cellular processes that involve highly curved structures. In the present study we examined the localization of endogenous synuclein in bovine chromaffin cells by immunocytochemistry and its possible function to control protein discharge upon fusion of the granule with the plasma membrane by regulating the fusion pore. We found with quantitative immunocytochemistry that endogenous ß-synuclein associates with secretory granules. Endogenous α-synuclein only rarely co-localizes with secretory granules. Overexpression of α-synuclein but not ß-synuclein quickened the post- fusion discharge of BDNF-pHluorin by approxinately 30%. However, neither α- nor ß-synuclein significantly altered curvature dynamics associated with fusion pore expansion that were measured by the combination of polarization and total internal reflection fluorescence microscopy (pTIRFM). Whatever the mechanism, the physiological significance of the small increased rate of post-fusion protein discharge caused by α-synuclein remains to be demonstrated, especially since endogenous ß-, but not α-synuclein is the predominant synuclein isoform associated with chromaffin granules.

Chromogranin A, the major lumenal protein in chromaffin granules, controls fusion pore expansion.

Upon fusion of the secretory granule with the plasma membrane, small molecules are discharged through the immediately formed narrow fusion pore, but protein discharge awaits pore expansion. Recently, fusion pore expansion was found to be regulated by tissue plasminogen activator (tPA), a protein present within the lumen of chromaffin granules in a subpopulation of chromaffin cells. Here, we further examined the influence of other lumenal proteins on fusion pore expansion, especially chromogranin A (CgA), the major and ubiquitous lumenal protein in chromaffin granules. Polarized TIRF microscopy demonstrated that the fusion pore curvature of granules containing CgA-EGFP was long lived, with curvature lifetimes comparable to those of tPA-EGFP-containing granules. This was surprising because fusion pore curvature durations of granules containing exogenous neuropeptide Y-EGFP (NPY-EGFP) are significantly shorter (80% lasting <1 s) than those containing CgA-EGFP, despite the anticipated expression of endogenous CgA. However, quantitative immunocytochemistry revealed that transiently expressed lumenal proteins, including NPY-EGFP, caused a down-regulation of endogenously expressed proteins, including CgA. Fusion pore curvature durations in nontransfected cells were significantly longer than those of granules containing overexpressed NPY but shorter than those associated with granules containing overexpressed tPA, CgA, or chromogranin B. Introduction of CgA to NPY-EGFP granules by coexpression converted the fusion pore from being transient to being longer lived, comparable to that found in nontransfected cells. These findings demonstrate that several endogenous chromaffin granule lumenal proteins are regulators of fusion pore expansion and that alteration of chromaffin granule contents affects fusion pore lifetimes. Importantly, the results indicate a new role for CgA. In addition to functioning as a prohormone, CgA plays an important role in controlling fusion pore expansion.

Slow fusion pore expansion creates a unique reaction chamber for co-packaged cargo.

A lumenal secretory granule protein, tissue plasminogen activator (tPA), greatly slows fusion pore dilation and thereby slows its own discharge. We investigated another outcome of the long-lived narrow fusion pore: the creation of a nanoscale chemical reaction chamber for granule contents in which the pH is suddenly neutralized upon fusion. Bovine adrenal chromaffin cells endogenously express both tPA and its primary protein inhibitor, plasminogen activator inhibitor 1 (PAI). We found by immunocytochemistry that tPA and PAI are co-packaged in the same secretory granule. It is known that PAI irreversibly and covalently inactivates tPA at neutral pH. We demonstrate with zymography that the acidic granule lumen protects tPA from inactivation by PAI. Immunocytochemistry, total internal reflection fluorescence (TIRF) microscopy, and polarized TIRF microscopy demonstrated that co-packaged PAI and tPA remain together in granules for many seconds in the nanoscale reaction chamber, more than enough time to inhibit tPA and create a new secreted protein species.

Refractive Index Imaging of Cells with Variable-Angle Near-Total Internal Reflection (TIR) Microscopy.

The refractive index in the interior of single cells affects the evanescent field depth in quantitative studies using total internal reflection (TIR) fluorescence, but often that index is not well known. We here present method to measure and spatially map the absolute index of refraction in a microscopic sample, by imaging a collimated light beam reflected from the substrate/buffer/cell interference at variable angles of incidence. Above the TIR critical angle (which is a strong function of refractive index), the reflection is 100%, but in the immediate sub-critical angle zone, the reflection intensity is a very strong ascending function of incidence angle. By analyzing the angular position of that edge at each location in the field of view, the local refractive index can be estimated. In addition, by analyzing the steepness of the edge, the distance-to-substrate can be determined. We apply the technique to liquid calibration samples, silica beads, cultured Chinese hamster ovary cells, and primary culture chromaffin cells. The optical technique suffers from decremented lateral resolution, scattering, and interference artifacts. However, it still provides reasonable results for both refractive index (~1.38) and for distance-to-substrate (~150 nm) for the cells, as well as a lateral resolution to about 1 µm.

Protein mobility within secretory granules.

We investigated the basis for previous observations that fluorescent-labeled neuropeptide Y (NPY) is usually released within 200 ms after fusion, whereas labeled tissue plasminogen activator (tPA) is often discharged over many seconds. We found that tPA and NPY are endogenously expressed in small and different subpopulations of bovine chromaffin cells in culture. We measured the mobility of these proteins (tagged with fluorophore) within the lumen of individual secretory granules in living chromaffin cells, and related their mobilities to postfusion release kinetics. A method was developed that is not limited by standard optical resolution, in which a bright flash of strongly decaying evanescent field (∼64 nm exponential decay constant) produced by total internal reflection (TIR) selectively bleaches cerulean-labeled protein proximal to the glass coverslip within individual granules. Fluorescence recovery occurred as unbleached protein from distal regions within the 300 nm granule diffused into the bleached proximal regions. The fractional bleaching of tPA-cerulean (tPA-cer) was greater when subsequently probed with TIR excitation than with epifluorescence, indicating that tPA-cer mobility was low. The almost equal NPY-cer bleaching when probed with TIR and epifluorescence indicated that NPY-cer equilibrated within the 300 ms bleach pulse, and therefore had a greater mobility than tPA-cer. TIR-fluorescence recovery after photobleaching revealed a significant recovery of tPA-cer (but not NPY-cer) fluorescence within several hundred milliseconds after bleaching. Numerical simulations, which take into account bleach duration, granule diameter, and the limited number of fluorophores in a granule, are consistent with tPA-cer being 100% mobile, with a diffusion coefficient of 2 × 10(-10) cm(2)/s (∼1/3000 of that for a protein of similar size in aqueous solution). However, the low diffusive mobility of tPA cannot alone explain its slow postfusion release. In the accompanying study, we suggest that, additionally, tPA itself stabilizes the fusion pore with dimensions that restrict its own exit.

Lumenal protein within secretory granules affects fusion pore expansion.

It is often assumed that upon fusion of the secretory granule membrane with the plasma membrane, lumenal contents are rapidly discharged and dispersed into the extracellular medium. Although this is the case for low-molecular-weight neurotransmitters and some proteins, there are numerous examples of the dispersal of a protein being delayed for many seconds after fusion. We have investigated the role of fusion-pore expansion in determining the contrasting discharge rates of fluorescent-tagged neuropeptide-Y (NPY) (within 200 ms) and tissue plasminogen activator (tPA) (over many seconds) in adrenal chromaffin cells. The endogenous proteins are expressed in separate chromaffin cell subpopulations. Fusion pore expansion was measured by two independent methods, orientation of a fluorescent probe within the plasma membrane using polarized total internal reflection fluorescence microscopy and amperometry of released catecholamine. Together, they probe the continuum of the fusion-pore duration, from milliseconds to many seconds after fusion. Polarized total internal reflection fluorescence microscopy revealed that 71% of the fusion events of tPA-cer-containing granules maintained curvature for >10 s, with approximately half of the structures likely connected to the plasma membrane by a short narrow neck. Such events were not commonly observed upon fusion of NPY-cer-containing granules. Amperometry revealed that the expression of tPA-green fluorescent protein (GFP) prolonged the duration of the prespike foot ∼2.5-fold compared to NPY-GFP-expressing cells and nontransfected cells, indicating that expansion of the initial fusion pore in tPA granules was delayed. The t1/2 of the main catecholamine spike was also increased, consistent with a prolonged delay of fusion-pore expansion. tPA added extracellularly bound to the lumenal surface of fused granules. We propose that tPA within the granule lumen controls its own discharge. Its intrinsic biochemistry determines not only its extracellular action but also the characteristics of its presentation to the extracellular milieu.

A nibbling mechanism for clathrin-mediated retrieval of secretory granule membrane after exocytosis.

Clathrin-mediated endocytosis is the major pathway for recycling of granule membrane components after strong stimulation and high exocytotic rates. It resembles "classical" receptor-mediated endocytosis but has a trigger that is unique to secretion, the sudden appearance of the secretory granule membrane in the plasma membrane. The spatial localization, the relationship to individual fusion events, the nature of the cargo, and the timing and nature of the nucleation events are unknown. Furthermore, a size mismatch between chromaffin granules (∼300-nm diameter) and typical clathrin-coated vesicles (∼90 nm) makes it unlikely that clathrin-mediated endocytosis internalizes as a unit the entire fused granule membrane. We have used a combination of total internal reflection fluorescence microscopy of transiently expressed proteins and time-resolved quantitative confocal imaging of endogenous proteins along with a fluid-phase marker to address these issues. We demonstrate that the fused granule membrane remains a distinct entity and serves as a nucleation site for clathrin- and dynamin-mediated endocytosis that internalizes granule membrane components in small increments.

Real-time imaging of plasma membrane deformations reveals pre-fusion membrane curvature changes and a role for dynamin in the regulation of fusion pore expansion.

Assays for real-time investigation of exocytosis typically measure what is released from the granule. From this, inferences are made about the dynamics of membrane remodeling as fusion progresses from start to finish. We have recently undertaken a different approach to investigate the fusion process, by focusing not primarily on the granule, but rather its partner in exocytosis - the plasma membrane. We have been guided by the idea that biochemical interactions between the granule and plasma membranes before and during fusion, cause changes in membrane conformation. To enable study of membrane conformation, a novel imaging technique was developed combining polarized excitation of an oriented membrane probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI) with total internal reflection fluorescence microscopy (pTIRFM). Because this technique measures changes in membrane conformation (or deformations) directly, its usefulness persists even after granule cargo reporter (catecholamine, or protein), is no longer present. In this mini-review, we first summarize the workings of pTIRFM. We then discuss the application of the technique to investigate deformations in the membrane preceding fusion, and later, during fusion pore expansion. Finally, we discuss how expansion of the fusion pore may be regulated by the GTPase activity of dynamin.

Subunit-dependent axonal trafficking of distinct alpha heteromeric potassium channel complexes.

Voltage-gated potassium (Kv) channels are critical for neuronal excitability and are targeted to specific subcellular compartments to carry out their unique functions. While it is widely believed that Kv channels exist as heteromeric complexes in neurons, direct tests of the hypothesis that specific heteromeric channel populations display divergent spatial and temporal dynamics are limited. Using a bimolecular fluorescence complementation approach, we monitored the assembly and localization of cell surface channel complexes in living cells. While PSD95-mediated clustering was subunit independent, selective visualization of heteromeric Kv complexes in rat hippocampal neurons revealed subunit-dependent localization that was not predicted by analyzing individual subunits. Assembly of Kv1.1 with Kv1.4 prevented axonal localization but not surface expression, while inclusion of Kv1.2 imparted clustering at presynaptic sites and decreased channel mobility within the axon. This mechanism by which specific Kv channel subunits can act in a dominant manner to impose unique trafficking properties to heteromeric complexes extended to Shab-related family of Kv channels. When coexpressed, Kv2.1 and Kv2.2 heteromultimers did not aggregate in somatodendritic clusters observed with expression of Kv2.1 alone. These studies demonstrate selective axonal trafficking and surface localization of distinct Kv channels based on their subunit composition.

A new role for the dynamin GTPase in the regulation of fusion pore expansion.

Dynamin is a master regulator of membrane fission in endocytosis. However, a function for dynamin immediately upon fusion has also been suspected from a variety of experiments that measured release of granule contents. The role of dynamin guanosine triphosphate hydrolase (GTPase) activity in controlling fusion pore expansion and postfusion granule membrane topology was investigated using polarization optics and total internal reflection fluorescence microscopy (pTIRFM) and amperometry. A dynamin-1 (Dyn1) mutant with increased GTPase activity resulted in transient deformations consistent with rapid fusion pore widening after exocytosis; a Dyn1 mutant with decreased activity slowed fusion pore widening by stabilizing postfusion granule membrane deformations. The experiments indicate that, in addition to its role in endocytosis, GTPase activity of dynamin regulates the rapidity of fusion pore expansion from tens of milliseconds to seconds after fusion. These findings expand the membrane-sculpting repertoire of dynamin to include the regulation of immediate postfusion events in exocytosis that control the rate of release of soluble granule contents.

Polarized TIRFM reveals changes in plasma membrane topology before and during granule fusion.

We have recently developed a combination of polarization and total internal reflection fluorescence microscopy (pTIRFM) to monitor changes in plasma membrane topology occurring after fusion of chromaffin granules. In this report, pTIRFM is further exploited to reveal two major findings in regards to the secretory pathway in bovine chromaffin cells. First, we show that changes in membrane topology are sometimes detected even prior to fusion. This occurs with high probability in a small subset of granules that appear in the evanescent field during the experiment. On these occasions, the plasma membrane invaginates with the movement just preceding the appearance of a granule in the evanescent field. Such events may represent a direct interaction of the granule with the plasma membrane. Second, we show that the topological fate of the post-fusion, granule/plasma membrane intermediate is regulated by divalent cation. When Sr2+ is used instead of Ca2+ to trigger exocytosis, membrane topology in the exocytotic region is stabilized with significant curvature and indentation.

Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM.

Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane-cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the postfusion granule membrane-plasma membrane complex. Experiments on diI-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin.