In order to understand exocytosis and endocytosis, it is necessary to study these processes directly. An elegant way to do this is by measuring plasma membrane capacitance (Cm), a parameter proportional to cell surface area, the fluctuations of which are due to fusion and fission of secretory and other vesicles. Here we describe protocols that enable high-resolution Cm measurements in macroscopic and microscopic modes. Macroscopic mode, performed in whole-cell configuration, is used for measuring bulk Cm changes in the entire membrane area, and it enables the introduction of exocytosis stimulators or inhibitors into the cytosol through the patch pipette. Microscopic mode, performed in cell-attached configuration, enables measurements of Cm with attofarad resolution and allows characterization of fusion pore properties. Although we usually apply these protocols to primary pituitary cells and astrocytes, they can be adapted and used for other cell types. After initial hardware setup and culture preparation, several Cm measurements can be performed daily.
COBISS.SI-ID: 30613977
Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication. The membrane fusion process is mediated by a conservedset of SNARE proteins: vesicuJar synaptobrevin and plasma membrane syntaxin and SNAP-25. Recent data suggest that the fusion process may be subject to regulation by local lipid metabolism. Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin. We show that sphingosine, areleasable backbone of sphingolipids, activates synaptobrevin in synaptic vesicles to form the SNARE compJex implicated in membrane fusion. Consistent with the roJe of synaptobrevin in vesicle fusion, sphingosine upregulated exocytosis in isolated nerve terminais, neuromuscular junctions, neuroendocrine celi s and hippocampal neurons, but not in neurons obtained from synaptobrevin-2 knockoutmice. Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining anovel function for this important lipid regulator.
COBISS.SI-ID: 25625305
While serotonin (5-HT) co-localization with insulin in granules of pancreatic ß-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is stili unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/-) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- ß-cells, which clearly showed that the secretory defect is downstream of Ca2+ -signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in ß-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic ß-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.
COBISS.SI-ID: 63941377
The release ofhormones and neurotransmitters, mediated by regulated exocytosis, can be modified by regulation of the fusion pore. The fusion pore is considered stable and narrow initially, eventually leading to the complete merger of the vesicle and the plasma membranes. By using the high- resolution patch -clamp capacitance technique, we studied single vesicles and asked whether the Secl/Muncl8 proteins, interacting with the membrane fusion-mediating SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, affect fusion pore properties. Muncl8-1 mutants were transfected into lactotrophs to affect the interaction of Muncl8-1 with syntaxinl (Synt1) (R39C), Rab3A (E466K), and Mints (P242S). Compared with wild-type, Muncl8-1 E466K increased the frequency ofthe fusion event. The latter two mutants increased the fusion pore dwell-time. All the mutants stabilized narrow fusion pores and increased the amplitude of fusion events, likely via preferential fusion oflarger vesicIes, since overexpression of Munc18-1 R39C did not affect the ave rage size of vesicIes, as determined by stimulated emission depletion (STED) microscopy. Single-molecule atomic force microscopy experiments revealed that wild-type Munc18-1, but not Munc18-1 R39C, abrogates the interaction between synaptobrevin2 (Syb2) and Synt1 binarytrans-complexes. However, neither form ofMuncl8-1 affected the interaction ofSyb2 with the preformed binary cis-Synt1ASNAP25B complexes. Thisindicates that Munc18-1 performs a proofing function by inhibiting tethering of Syb2-containing ves ici es solely to Synt1 at the plasmalemma andfavoring vesicular tethering to the preformed binary cis-complex of Synt1A-SNAP25B. The association of Muncl8-1 with the ternary SNARE complex leads to tuning of fusion pores via multiple and converging mechanisms involving Muncl8-1 interactions with Synt1A, Rab3A, and Mints.
COBISS.SI-ID: 28521433
Exocytic transmitter release is regulated by the SNARE complex, which contains a vesicular protein, synaptobrevin2 (Sb2). However, Sb2 vesicular arrangement is unclear. Here we use super-resolution fluorescence microscopy to study the prevalence and distribution of endogenous and exogenous Sb2 in single vesicles of astrocytes, the most abundant glial cells in the brain. We tag Sb2 protein at C- and N termini with a pair of fluorophores, which allows us to determine the Sb2 length and geometry. To estimate total number of Sb2 proteins per vesicle and the quantity necessary for the formation of fusion pores, we treat cells with ATP to stimulate Ca2+-dependent exocytosis, increase intracellular alkalinity to enhance the fluorescence presentation of yellow-shifted pHluorin (YpH), appended to the vesicle lumen domain of Sb2, and perform photobleaching of YpH fluorophores. Fluorescence intensity analysis reveals that the total number of endogenous Sb2 units or molecules per vesicle is (-25.
COBISS.SI-ID: 31384793