Large Dense-core Secretory Vesicles Research Articles

A conserved electrostatic membrane-binding surface in synaptotagmin-like proteins revealed using molecular phylogenetic analysis and homology modeling.

Protein structure prediction has emerged as a core technology for understanding biomolecules and their interactions. Here, we combine homology-based structure prediction with molecular phylogenetic analysis to study the evolution of electrostatic membrane binding among the vertebrate synaptotagmin-like protein (Slp) family. Slp family proteins play key roles in the membrane trafficking of large dense-core secretory vesicles. Our previous experimental and computational study found that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to anionic phospholipids in the cytoplasmic leaflet of the plasma membrane through a large positively charged protein surface centered on a cluster of phosphoinositide-binding lysine residues. Because this surface contributes greatly to Slp-4 C2A domain membrane binding, we hypothesized that the net charge on the surface might be evolutionarily conserved. To test this hypothesis, the known C2A sequences of Slp-4 among vertebrates were organized by class (from mammalia to pisces) using molecular phylogenetic analysis. Consensus sequences for each class were then identified and used to generate homology structures, from which Poisson-Boltzmann electrostatic potentials were calculated. For comparison, homology structures and electrostatic potentials were also calculated for the five human Slp protein family members. The results demonstrate that the charge on the membrane-binding surface is highly conserved throughout the evolution of Slp-4, and more highly conserved than many individual residues among the human Slp family paralogs. Such molecular phylogenetic-driven computational analysis can help to describe the evolution of electrostatic interactions between proteins and membranes which are crucial for their function.

Using protein structure prediction with molecular phylogenetic analysis to understand membrane interactions in synaptotagmin-like proteins

Protein structure prediction is emerging as a core technology for understanding biomolecules and their interactions. Here, we have combined structure prediction with molecular phylogenetic analysis in order to provide information on the evolution of electrostatic membrane binding in synaptotagmin-like proteins (Slps). Slp family proteins play key roles in the membrane trafficking of large dense-core secretory vesicles in vertebrates. Our previous experimental and computational study found that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylserine (PS) found in the cytoplasmic leaflet of the plasma membrane.

Structure Prediction and Molecular Phylogenetic Analysis of Membrane Interactions in Synaptotagmin-Like Proteins

Membrane trafficking and exocytosis are evolutionarily conserved throughout eukaryotes and involve many interactions between proteins and membrane surfaces. In vertebrates, synaptotagmin-like proteins (Slp) play key roles in trafficking of large dense-core secretory vesicles. Slp family proteins promote secretory vesicle docking to the plasma membrane through their N-terminal Slp homology domains, which bind to vesicular Rab proteins, and their tandem C-terminal C2 domains (C2A and C2B), which bind plasma membrane lipids independently of calcium. Our previous experimental and computational studies showed that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to membrane surfaces containing phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylserine (PS). A conserved PIP2 binding site centered on three lysine residues in the beta3-beta4 region specifically anchors the domain to PIP2 lipids, while many other basic residues surrounding the Lys-cluster enhance interactions with PS. We hypothesize that the positive electrostatic potential of the membrane binding surface is a key determinant of its membrane affinity and function, and therefore the net charge on the protein surface is likely to be evolutionarily conserved. To test this hypothesis, the C2A domain sequences for Slp-4 and Slp-2 in vertebrates were compared to assess the evolution of their respective polybasic surfaces. Using a molecular phylogenetic approach, clades were identified and the net charges of individual and consensus sequences were calculated. Structural homology models were generated using a crystal structure of Slp-4 C2A, and Poisson-Boltzmann calculations were carried out to quantify the positive electrostatic surfaces. This approach may help characterize electrostatic interactions with charged membrane surfaces and identify conserved differences that govern affinity and function of membrane-binding proteins.

Multivalent lipid targeting by the calcium-independent C2A domain of synaptotagmin-like protein 4/granuphilin

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine but much weaker when either the background anionic lipids or PIP2 is removed. Through computational and experimental approaches, we show that this high-affinity membrane binding arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, a larger cationic surface surrounding the cluster contributes substantially to the affinity for physiologically relevant lipid compositions. Although the K398A mutation in the lysine cluster blocks PIP2 binding, this mutated protein domain retains the ability to bind physiological membranes in both a liposome-binding assay and MIN6 cells. Molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant that arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and binds weakly to membranes. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high-affinity docking of large dense core secretory vesicles to the plasma membrane.

A rare case of pancreatic neuroendocrine tumor

Pancreatic neuroendocrine tumors (PNETs) are a group of endocrine neoplasms that exhibit neuroendocrine phenotypes. These include the production of neuropeptides, large dense-core secretory vesicles, and a lack of neural structures. They arise in the pancreas and are among the most common neuroendocrine tumors. A case of 38 years old female presented with a painless progressive swelling in the right upper abdomen since one month which is insidious in onset, gradually increasing in size with a history of significant weight loss and patient is not a known diabetic. On Examination, a swelling of size 5x7cm noted involving epigastric and right hypochondrium, which is not moving with respiration and dull on percussion. Plane of swelling is Intra-abdominal& retroperitoneal. Computed tomography abdomen (CECT) showed well defined rounded iso dense lesion showing heterogeneous enhancement in arterial and venous phase. Magnetic resonance cholangiopancreatography (MRCP) showed well defined lobulated heterogeneous solid lesion with central areas of necrosis seen in the pancreaticoduodenal groove (PDG) involving main pancreatic duct (MPD) and loss of fat planes with surrounding structures most likely malignant etiology. The patient underwent pancreaticoduodenectomy surgery (Whipple procedure) and had an uneventful recovery. Post-operative biopsy report confirmed pancreatic neuroendocrine tumor grade II (well-differentiated type), stage II a – pT3 N0 Mx.

Membrane Binding of Synaptotagmin-Like Protein 4: Insight from Molecular Dynamics Simulations

Synaptotagmin-like proteins (Slps) are a family of Rab GTPase effectors involved in secretory vesicle trafficking and membrane docking. Slps possess an N-terminal domain that binds Rab27 on large dense-core secretory vesicles and one or two C-terminal C2 domains that dock to plasma membranes via interaction with anionic lipid molecules such as phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylserine (PS). In particular, Slp-4, also known as granuphilin, docks secretory vesicles to the plasma membrane primarily via its C2A domain, which has a high affinity for anionic lipid bilayers. Although the structure of the Slp-4 C2A domain is known and some of its key PIP2-binding residues have been identified, the full structural mechanism of its strong membrane binding remains unclear. Here, we employ molecular dynamics simulations of the Slp-4 C2A domain to determine contributions of particular residues in binding to a lipid bilayer containing physiologically relevant levels of the anionic target lipids PS and PIP2. Simulations include mutations of two important residues: Lys398, which is known to participate in PIP2 coordination, and Phe452, which is suspected to insert into target membranes hydrophobically. Preliminary results support the experimental finding that both the K398A and F452A mutants are capable of binding membranes but suggest a different balance of lipid interactions in the two mutants. Overall, experimental and computational results indicate that high-affinity membrane binding of the Slp-4 C2A domain is sustained via many lipid-interacting residues distributed over a large electropositive protein surface.

Multivalent Membrane Lipid Targeting by the Calcium-Independent C2 Domains of Granuphilin: Evidence from Computation and Experiment

Granuphilin, also known as synaptotagmin-like protein 4 (Slp4), is a Rab effector responsible for docking insulin secretory vesicles to the plasma membrane prior to exocytosis. Granuphilin binds vesicular Rab proteins via an N-terminal Slp homology (SHD) domain, binds plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). Granuphilin's C2A domain has previously been shown to bind PIP2 or its soluble analogues with low micromolar affinity; however, the domain docks with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine (PS). Here we show using a combination of computational and experimental approaches that this high-affinity membrane interaction arises from concerted binding at two or more sites on the C2A domain. First, docking calculations predict at least two possible binding pockets for anionic ligands. Second, liposome binding measurements indicate that affinity depends on concentrations of PIP2 as well as PS and phosphatidic acid (PA) in the membrane. Third, fluorescence measurements indicate that different regions of the protein surface are responsible for binding membranes containing or lacking PIP2. Fourth, single-molecule lateral diffusion measurements indicate distinct membrane-bound states depending on the available target lipids. Mutational analysis is currently underway to confirm locations of these lipid binding sites. Overall, multivalent membrane binding by granuphilin likely contributes to selective recognition and high affinity docking of large dense-core secretory vesicles to the plasma membrane.

Research Resource: A Chromogranin A Reporter for Serotonin and Histamine Secreting Enteroendocrine Cells.

Chromogranin A (ChgA) is an acidic protein found in large dense-core secretory vesicles and generally considered to be expressed in all enteroendocrine cells of the gastrointestinal (GI) tract. Here, we characterize a novel reporter mouse for ChgA, ChgA-humanized Renilla reniformis (hr)GFP. The hrGFP reporter was found in the monoamine-storing chromaffin cells of the adrenal medulla, where ChgA was originally discovered. hrGFP also was expressed in enteroendocrine cells throughout the GI tract, faithfully after the expression of ChgA, as characterized by immunohistochemistry and quantitative PCR analysis of fluorescence-activated cell sorting-purified cells, although the expression in the small intestine was weak compared with that of the stomach and colon. In the stomach, hrGFP was highly expressed in almost all histamine-storing enterochromaffin (EC)-like cells, at a lower level in the majority of serotonin-storing EC cells and ghrelin cells, in a small fraction of somatostatin cells, but was absent from gastrin cells. In the small intestine, the hrGFP reporter was selectively, but weakly expressed in EC cells, although not in any peptide-storing enteroendocrine cells. In the colon, hrGFP was exclusively expressed in EC cells but absent from the peptide-storing enteroendocrine cells. In contrast, in the pancreas, hrGFP was expressed in β-cells, α-cells, and a fraction of pancreatic polypeptide cells. It is concluded that ChgA-hrGFP in the GI tract functions as an effective reporter, particularly for the large populations of still poorly characterized monoamine-storing enteroendocrine cells. Furthermore, our findings substantiate the potential function of ChgA as a monoamine-binding protein that facilitates the regulated endocrine secretion of large amounts of monoamines from enteroendocrine cells.

Peptidergic cell-specific synaptotagmins in Drosophila: localization to dense-core granules and regulation by the bHLH protein DIMMED.

Bioactive peptides are packaged in large dense-core secretory vesicles, which mediate regulated secretion by exocytosis. In a variety of tissues, the regulated release of neurotransmitters and hormones is dependent on calcium levels and controlled by vesicle-associated synaptotagmin (SYT) proteins. Drosophila express seven SYT isoforms, of which two (SYT-α and SYT-β) were previously found to be enriched in neuroendocrine cells. Here we show that SYT-α and SYT-β tissue expression patterns are similar, though not identical. Furthermore, both display significant overlap with the bHLH transcription factor DIMM, a known neuroendocrine (NE) regulator. RNAi-mediated knockdown indicates that both SYT-α and SYT-β functions are essential in identified NE cells as these manipulations phenocopy loss-of-function states for the indicated peptide hormones. In Drosophila cell culture, both SYT-α and neuropeptide cargo form DIMM-dependent fluorescent puncta that are coassociated by super-resolution microscopy. DIMM is required to maintain SYT-α and SYT-β protein levels in DIMM-expressing cells in vivo. In neurons normally lacking all three proteins (DIMM(-)/SYT-α(-)/SYT-β(-)), DIMM misexpression conferred accumulation of endogenous SYT-α and SYT-β proteins. Furthermore transgenic SYT-α does not appreciably accumulate in nonpeptidergic neurons in vivo but does so if DIMM is comisexpressed. Among Drosophila syt genes, only syt-α and syt-β RNA levels are upregulated by DIMM overexpression. Together, these data suggest that SYT-α and SYT-β are important for NE cell physiology, that one or both are integral membrane components of the large dense-core vesicles, and that they are closely regulated by DIMM at a post-transcriptional level.

Complexin activates exocytosis of distinct secretory vesicles controlled by different synaptotagmins.

Complexins are SNARE-complex binding proteins essential for the Ca(2+)-triggered exocytosis mediated by synaptotagmin-1, -2, -7, or -9, but the possible role of complexins in other types of exocytosis controlled by other synaptotagmin isoforms remains unclear. Here we show that, in mouse olfactory bulb neurons, synaptotagmin-1 localizes to synaptic vesicles and to large dense-core secretory vesicles as reported previously, whereas synaptotagmin-10 localizes to a distinct class of peptidergic secretory vesicles containing IGF-1. Both synaptotagmin-1-dependent synaptic vesicle exocytosis and synaptotagmin-10-dependent IGF-1 exocytosis were severely impaired by knockdown of complexins, demonstrating that complexin acts as a cofactor for both synaptotagmin-1 and synaptotagmin-10 despite the functional differences between these synaptotagmins. Rescue experiments revealed that only the activating but not the clamping function of complexins was required for IGF-1 exocytosis controlled by synaptotagmin-10. Thus, our data indicate that complexins are essential for activation of multiple types of Ca(2+)-induced exocytosis that are regulated by different synaptotagmin isoforms. These results suggest that different types of regulated exocytosis are mediated by similar synaptotagmin-dependent fusion mechanisms, that particular synaptotagmin isoforms confer specificity onto different types of regulated exocytosis, and that complexins serve as universal synaptotagmin adaptors for all of these types of exocytosis independent of which synaptotagmin isoform is involved.

The Functional Role of Chromogranins in Exocytosis

Chromogranins A (CgA) and B (CgB) are the main soluble proteins of large dense-core secretory vesicles (LDCVs). Using CgA- and CgB-knockout (KO) mice, we found that the absence of chromogranins A and B induces significant changes in catecholamine (CA) accumulation and the kinetics of exocytosis. By crossing these two knockout strains, we generated a viable and fertile double CgA/B-KO mouse in which the catecholamine content in chromaffin LDCVs was halved, and the secretory response significantly reduced. Incubating cells with L-DOPA increased the vesicular CA content in wild-type (WT) but not in Cg-KO cells, which was not due to changes in amine transport, or in the synthesis or degradation of cytosolic amines. Electron microscopy revealed the presence of giant secretory vesicles exhibiting significant alterations, with little or no electrodense inner matrix. Proteomic analysis confirmed the absence of CgA and B, and revealed small changes in SgII in the LDCV-enriched fraction, as well as the overexpression of fibrinogen and other proteins. In summary, our findings indicate that the mechanisms responsible for vesicular accumulation of CA are saturated in Cgs-KO cells, in contrast to the ample capacity for further accumulation in WT cells. We conclude that Cgs contribute to a highly efficient system that directly mediates monoamine accumulation and exocytosis in LDCVs.

Chromogranins A and B are key proteins in amine accumulation, but the catecholamine secretory pathway is conserved without them

Chromogranins are the main soluble proteins in the large dense core secretory vesicles (LDCVs) found in aminergic neurons and chromaffin cells. We recently demonstrated that chromogranins A and B each regulate the concentration of adrenaline in chromaffin granules and its exocytosis. Here we have further studied the role played by these proteins by generating mice lacking both chromogranins. Surprisingly, these animals are both viable and fertile. Although chromogranins are thought to be essential for their biogenesis, LDCVs were evident in these mice. These vesicles do have a somewhat atypical appearance and larger size. Despite their increased size, single-cell amperometry recordings from chromaffin cells showed that the amine content in these vesicles is reduced by half. These data demonstrate that although chromogranins regulate the amine concentration in LDCVs, they are not completely essential, and other proteins unrelated to neurosecretion, such as fibrinogen, might compensate for their loss to ensure that vesicles are generated and the secretory pathway conserved.

Spatial Distribution and Dynamics of Syntaxin-1 in Live PC-12 Cells

The soluble N-ethyl-maleimide-sensitive factor-attachment protein receptor (SNARE) proteins play an important role in vesicle fusion. The spatial distribution of these proteins in live cells is largely unknown. Clusters of syntaxin-1 (Syx-1) were observed previously by Hell, Lang and co-workers using stimulated emission depletion (STED) on PC-12 cell membrane sheets with immunostaining.1 The clusters may be docking sites for large dense-core secretory vesicles (LDCVs), which can then undergo exocytosis. In this study we transfected rat pheochromocytoma (PC-12) cells with a plasmid encoding Syx-1 tagged with the photoactivatable protein mEos2 at the N-terminus. Photoactivation and localization of single molecules was used to study the distribution of Syx-1 in live PC12 cells, while single particle tracking (SPT) was used to study the dynamics. The data indicate inhomogeneous diffusion, with some Syx-1 molecules appearing confined and others seemingly diffusing freely. A plot of is initially linear with a slope corresponding to a free diffusion constant of ∼0.15 µm2/sec. The spatial distribution is somewhat clustered, but seemingly much less so than in the earlier study of unroofed cells. LDCVs were imaged in sequence with Syx-1 using EGFP labeled tissue plasminogen activator (tPA). Preliminary data suggest that LDVCs visit the plasma membrane along specific branched paths whose locations tend to be anti-correlated with the distribution of Syx-1. 1JJ Sieber, et al. Science 317, 1072–1076 (2007).

Chromogranin B Gene Ablation Reduces the Catecholamine Cargo and Decelerates Exocytosis in Chromaffin Secretory Vesicles

Chromogranins/secretogranins (Cgs) are the major soluble proteins of large dense-core secretory vesicles (LDCVs). We have recently reported that the absence of chromogranin A (CgA) caused important changes in the accumulation and in the exocytosis of catecholamines (CAs) using a CgA-knock-out (CgA-KO) mouse. Here, we have analyzed a CgB-KO mouse strain that can be maintained in homozygosis. These mice have 36% less adrenomedullary epinephrine when compared to Chgb(+/+) [wild type (WT)], whereas the norepinephrine content was similar. The total evoked release of CA was 33% lower than WT mice. This decrease was not due to a lower frequency of exocytotic events but to less secretion per quantum (approximately 30%) measured by amperometry; amperometric spikes exhibited a slower ascending but a normal decaying phase. Cell incubation with L-DOPA increased the vesicle CA content of WT but not of the CgB-KO cells. Intracellular electrochemistry, using patch amperometry, showed that L-DOPA overload produced a significantly larger increase in cytosolic CAs in cells from the KO animals than chromaffin cells from the WT. These data indicate that the mechanisms for vesicular accumulation of CAs in the CgB-KO cells were saturated, while there was ample capacity for further accumulation in WT cells. Protein analysis of LDCVs showed the overexpression of CgA as well as other proteins apparently unrelated to the secretory process. We conclude that CgB, like CgA, is a highly efficient system directly involved in monoamine accumulation and in the kinetics of exocytosis from LDCVs.

Synthesis of fusogenic lipids through activation of phospholipase D1 by GTPases and the kinase RSK2 is required for calcium-regulated exocytosis in neuroendocrine cells

Exocytosis of hormones occurs through the fusion of large dense-core secretory vesicles with the plasma membrane. This highly regulated process involves key proteins such as SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) and also specific lipids at the site of membrane fusion. Among the different lipids required for exocytosis, our recent observations have highlighted the crucial role of PA (phosphatidic acid) in the late stages of membrane fusion in various exocytotic events. An RNAi (RNA interference) strategy coupled with the detection of PA in living cells has pointed to plasma membrane-associated PLD1 (phospholipase D(1)) as the main producer of PA in response to secretagogue stimulation. We have identified several GTPases which regulate the activation level of PLD(1) in neuroendocrine cells. Finally, RSK2 (ribosomal S6 kinase 2) appears to phosphorylate and regulate the activity of PLD(1) in a calcium-dependent manner. Altogether our results have unravelled a complex set of regulatory pathways controlling the synthesis of fusogenic lipids at the secretory granule fusion site by PLD(1).

Three‐dimensional properties of GnRH neuroterminals in the median eminence of young and old rats

The decapeptide gonadotropin-releasing hormone (GnRH), which regulates reproduction in all vertebrates, is stored in, and secreted from, large dense-core secretory vesicles in nerve terminals in the median eminence. GnRH is released from these terminals with biological rhythms that are critical for the maintenance of normal reproduction. During reproductive aging in female rats, there is a loss of GnRH pulses and a diminution of the GnRH surge. However, information about the specific role of GnRH nerve terminals is lacking, particularly in the context of aging. We sought to gain novel ultrastructural information about GnRH neuroterminals by performing three-dimensional (3D) reconstructions of GnRH neuroterminals and their surrounding microenvironment in the median eminence of young (4-5 months) and old (22-24 months) ovariectomized Sprague-Dawley female rats. Median eminence tissues were freeze-plunge embedded and serial ultrathin sections were collected on slot grids for immunogold labeling of GnRH immunoreactivity. Sequential images were used to create 3D models of GnRH terminals. These reconstructions provided novel perspectives into the morphological properties of GnRH terminals and their neural and glial environment. We also noted that the cytoarchitectural features of the median eminence became disorganized with aging. Quantitative measures showed a significant decrease in the apposition between GnRH terminal membranes and glial cells. Our data suggest reproductive aging in rats is characterized by structural organizational changes to the GnRH terminal microenvironment in the median eminence.

Fluorescent Cargo Proteins in Peptidergic Endocrine Cells

Fluorescent fusion proteins are an important tool for the study of vesicle trafficking and exocytosis, especially when combined with newer types of microscopy. We previously reported that the design of a vesicle-targeted fluorescent fusion construct strongly influences the kinetics of fluorescence change at exocytosis. In the present study we demonstrate that the cell in which a construct is expressed also affects the kinetics of fluorescence change at exocytosis. We fused enhanced green fluorescent protein to the carboxy terminus of the vesicular cargo protein rodent islet amyloid polypeptide. The two proteins were separated by a "linker" sequence of 18 amino acids. We then compared kinetics of fluorescence change at exocytosis for this fluorescent cargo protein expressed in three different types of peptidergic endocrine cell: pancreatic alpha cell, pancreatic beta cell, and adrenal chromaffin cell. In resting cells of all three types, fluorescent spots of similar size and membrane-proximal density appeared near the plasma membrane as expected if the probe is stored in large dense-core secretory vesicles. Upon stimulation, the fluorescent spots displayed sudden changes in fluorescence intensity that were consistent with exocytosis. In beta and alpha cells the fluorescent spots consistently brightened and persisted, whereas in chromaffin cells the fluorescent spots always dispersed rapidly. Thus, for fluorescent cargo proteins in peptidergic endocrine cells, cell type influences the kinetics of fluorescence change at exocytosis. Together with our previous findings, this observation strongly highlights the fact that the behavior of vesicle-targeted fluorescent cargo may be unrelated to that of native cargo, and it emphasizes the need for caution in interpreting fluorescence kinetics in terms of an exocytosis mechanism.

Heterogeneous Motion of Secretory Vesicles in the Actin Cortex of Live Cells: 3D Tracking to 5-nm Accuracy

Fluorescence microscopy tracks the three-dimensional motion of green fluorescent protein (GFP)-labeled large dense-core secretory vesicles (LDCVs) within the actin cortex of live PC12 cells. In this study, we achieve a 26-ms time resolution and a spatial accuracy of 5 nm or better in each dimension (one standard deviation in one dimension, σ1D). The resulting high-resolution trajectories reveal not only heterogeneity among vesicles but also heterogeneity within single-vesicle trajectories. As in earlier work, we observe three apparent groups of vesicles: the immobile, mobile, and directed-motion groups, but the distinctions among the groups are blurred. The directed trajectories exhibit segments with kinesin-like speed punctuated by pauses and changes in speed and direction. The immobile vesicles nearest the plasma membrane jump among sub-25-nm-diameter “mini-traps”. Comparison with microrheological data from entangled F-actin solutions suggests that the jumps may be caused by local remodeling of F-actin. Motion within a mini-trap is quantitatively modeled by a random walk in a parabolic restoring potential to yield single-trap restoring force constants of ∼0.04 pN/nm. As judged by mean-square displacement versus time, the mobile vesicles execute nearly free random walks in an elastic medium. We find no clear evidence of quasi-linear, directed motion in the mobile group. However, heterogeneity is evident in the distribution of frame-to-frame displacements, P(r), which requires a two-component fit. Evidently, mobile vesicles move by a combination of diffusion and motor-driven motion, with the direction changing rapidly as myosin-V crisscrosses the dense F-actin meshwork. The frequency of long frame-to-frame displacements of 25−70 nm suggests the presence of one or more myosin-V motors on the mobile vesicles. We argue that the motors on the immobile vesicles are less active or completely inactive. This suggests a regulatory mechanism for motor activity that may be related to the cell's ability to mobilize vesicles upon stimulation by Ca2+.

Exocytosis studies in a chromaffin cell-free system: imaging of single-vesicle exocytosis in a chromaffin cell-free system using total internal reflection fluorescence microscopy.

We have developed a system for the real-time study of regulated exocytosis in living, cultured bovine adrenal chromaffin cells (BCCs). Exocytosis was monitored by the use of total internal reflection fluorescence (TIRF) microscopy to image single large dense-core secretory vesicles (LDCVs). Fluorescent labeling of LDCVs was achieved either with the membrane-permeant weak base, acridine orange (AO), or by transduction of BCCs so as to express a fluorescent chimeric "cargo" protein that is targeted to LDCVs. In either case, exocytosis is visible by the disappearance of a vesicle accompanied by a bright flash as the fluorescent contents leave the acidic LDCV lumen, move towards the source of the evanescent wave, and disperse into the extracellular medium. Furthermore, for the first time, we have developed a broken-cell system for real-time imaging in BCCs, in which individual plated cells are mechanically "unroofed" with a jet of intracellular medium, leaving a membrane patch with docked vesicles on the coverslip. In this cell-free system, a subpopulation of docked granules undergoes exocytosis in response to calcium. This approach allows us direct experimental access to membrane-docked LDCVs in order to investigate the dependence of exocytosis on defined protein components and intracellular conditions at the single-vesicle level. In addition, this system can be used for a reconstitution analysis of the exocytosis machinery. Finally, we demonstrate the use of 2D+1 image analysis for visualizing single-vesicle exocytosis. We use this approach for a rapid analysis of larger numbers of imaged vesicles.

Lithium Ions Up‐Regulate mRNAs Encoding Dense‐Core Vesicle Proteins in Nerve Growth Factor‐Differentiated PC12 Cells

We recently reported that lithium ions induced an up-regulation of cysteine string protein (CSP) gene expression in nerve growth factor (NGF)-differentiated PC12 cells but not in undifferentiated cells. Concomitantly, expression of two other proteins of regulated secretory pathways, synaptophysin (SY) and SNAP-25, was unaffected by lithium. To assess further the specificity of this effect of lithium, we used cDNA arrays. Our data indicate that lithium ions increase the level of mRNA for proteins such as secretogranin II and vesicular monoamine transporter 1 that are preferentially associated with large densecore secretory vesicles (LDCVs) without affecting mRNAs for proteins predominantly affiliated with small synaptic-like vesicles, including the vesicular acetylcholine transporter and SY. This action of lithium is detected in NGF-differentiated PC12 cells but not in undifferentiated cells. These observations suggest that lithium ions modulate the turnover of LDCVs, and this may play a role in mediating the therapeutic action of lithium in manic-depressive illness.