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Tissue-specific autoimmunity occurs when selected antigens presented by susceptible alleles of the major histocompatibility complex are recognized by T cells. However, the reason why certain specific self-antigens dominate the response and are indispensable for triggering autoreactivity is unclear. Spontaneous presentation of insulin is essential for initiating autoimmune type 1 diabetes in non-obese diabetic mice.sup.1,2. A major set of pathogenic CD4 T cells specifically recognizes the 12-20 segment of the insulin B-chain (B:12-20), an epitope that is generated from direct presentation of insulin peptides by antigen-presenting cells.sup.3,4. These T cells do not respond to antigen-presenting cells that have taken up insulin that, after processing, leads to presentation of a different segment representing a one-residue shift, B:13-21.sup.4. CD4 T cells that recognize B:12-20 escape negative selection in the thymus and cause diabetes, whereas those that recognize B:13-21 have only a minor role in autoimmunity.sup.3-5. Although presentation of B:12-20 is evident in the islets.sup.3,6, insulin-specific germinal centres can be formed in various lymphoid tissues, suggesting that insulin presentation is widespread.sup.7,8. Here we use live imaging to document the distribution of insulin recognition by CD4 T cells throughout various lymph nodes. Furthermore, we identify catabolized insulin peptide fragments containing defined pathogenic epitopes in [beta]-cell granules from mice and humans. Upon glucose challenge, these fragments are released into the circulation and are recognized by CD4 T cells, leading to an activation state that results in transcriptional reprogramming and enhanced diabetogenicity. Therefore, a tissue such as pancreatic islets, by releasing catabolized products, imposes a constant threat to self-tolerance. These findings reveal a self-recognition pathway underlying a primary autoantigen and provide a foundation for assessing antigenic targets that precipitate pathogenic outcomes by systemically sensitizing lymphoid tissues.A sensitive T cell tracking assay reveals immunogenic activity of specific catabolized peptide fragments of insulin and their effects on T cell activity in lymph nodes, highlighting communication between pancreatic islets and lymphoid tissue., Author(s): Xiaoxiao Wan [sup.1] , Bernd H. Zinselmeyer [sup.1] , Pavel N. Zakharov [sup.1] , Anthony N. Vomund [sup.1] , Ruth Taniguchi [sup.2] , Laura Santambrogio [sup.3] , Mark S. [...]

Rapid plasma membrane resealing is essential for cellular survival. Earlier studies showed that plasma membrane repair requires [Ca.sup.2+]-dependent exocytosis of lysosomes and a rapid form of endocytosis that removes membrane lesions. However, the functional relationship between lysosomal exocytosis and the rapid endocytosis that follows membrane injury is unknown. In this study, we show that the lysosomal enzyme acid sphingomyelinase (ASM) is released extracellularly when cells are wounded in the presence of [Ca.sup.2+]. ASM-deficient cells, including human cells from Niemann-Pick type A (NPA) patients, undergo lysosomal exocytosis after wounding but are defective in injury-dependent endocytosis and plasma membrane repair. Exogenously added recombinant human ASM restores endocytosis and resealing in ASM-depleted cells, suggesting that conversion of plasma membrane sphingomyelin to ceramide by this lysosomal enzyme promotes lesion internalization. These findings reveal a molecular mechanism for restoration of plasma membrane integrity through exocytosis of lysosomes and identify defective plasma membrane repair as a possible component of the severe pathology observed in NPA patients. doi/10.1083/jcb.201003053

Surface-patterned platinum microelectrodes insulated with 300 nm thick fused silica were fabricated using contact photolithography. These electrodes exhibit low noise and were used for monitoring single vesicle exocytosis from chromaffin cells by constant potential amperometry as well as fast-scan cyclic voltammetry. Amperometric spike parameters were consistent with those obtained with conventional carbon fiber electrodes. Catecholamine voltammograms acquired with platinum electrodes exhibited redox peaks with full width at half-maximum of ~45 mV, much sharper than those of carbon fiber electrode recordings. The time course of voltammetrically measured release events was similar for platinum and carbon fiber electrodes. The fused-silica-insulated platinum electrodes could be cleaned and reused repetitively and allowed incorporation of micrometer precision surface-patterned poly-D-lysine. Poly-D-lysine-functionalized devices were applied to stimulate mast cells and record single release events without serotonin preloading. Microfabricated platinum electrodes are thus able to record single exocytotic events with high resolution and should be suitable for highly parallel electrode arrays allowing simultaneous measurements of single events from multiple cells. 10.1021/ac900674g

The time at which the N-ethylmaleimide-sensitive factor (NSF) acts during synaptic vesicle (SV) trafficking was identified by time-controlled perturbation of NSF function with a photoactivatable inhibitory peptide. Photolysis of this caged peptide in the squid giant presynaptic terminal caused an abrupt (0.2 s) slowing of the kinetics of the postsynaptic current (PSC) and a more gradual (2-3 s) reduction in PSC amplitude. Based on the rapid rate of these inhibitory effects relative to the speed of SV recycling, we conclude that NSF functions in reactions that immediately precede neurotransmitter release. Our results indicate the locus of SNARE protein recycling in presynaptic terminals and reveal NSF as a potential target for rapid regulation of transmitter release. caged probes | exocytosis | synaptic transmission | synaptic vesicle cycle

At the first synapse in the auditory pathway, the receptor potential of mechanosensory hair cells is converted into a firing pattern in auditory nerve fibers. For the accurate coding of timing and intensity of sound signals, transmitter release at this synapse must occur with the highest precision. To measure directly the transfer characteristics of the hair cell afferent synapse, we implemented simultaneous whole-cell recordings from mammalian inner hair cells (IHCs) and auditory nerve fiber terminals that typically receive input from a single ribbon synapse. During a 1-s IHC depolarization, the synaptic response depressed >90%, representing the main source for adaptation in the auditory nerve. Synaptic depression was slightly affected by [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor desensitization; however, it was mostly caused by reduced vesicular release. When the transfer function between transmitter release and [Ca.sup.2+] influx was tested at constant open probability for [Ca.sup.2+] channels (potentials >0 mV), a super linear relation was found. This relation is presumed to result from the cooperative binding of three to four [Ca.sup.2+] ions at the [Ca.sup.2+] sensor. However, in the physiological range for receptor potentials (-50 to -30 mV), the relation between [Ca.sup.2+] influx and afferent activity was linear, assuring minimal distortion in the coding of sound intensity. Changes in [Ca.sup.2+] influx caused an increase in release probability, but not in the average size of multivesicular synaptic events. By varying [Ca.sup.2+] buffering in the IHC, we further investigate how [Ca.sup.2+] channel and [Ca.sup.2+] sensor at this synapse might relate. hearing | exocytosis | hair cell | synaptic transmission | auditory nerve fiber

The state of secretory granules immediately before fusion with the plasma membrane is unknown, although the granules are generally assumed to be stably bound (docked). We had previously developed methods using total internal reflection fluorescence microscopy and image analysis to determine the position of chromaffin granules immediately adjacent to the plasma membrane with high precision, often to within [approximately equal to] 10 nm, or chromaffin cells | exocytosis | granule motion | total internal reflection fluorescence microscopy

The effects of intracellular [Cl.sup.-] concentration ([[[Cl.sup.-]].sup.i]) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated [Ca.sup.2+]-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 [micro]M) or a [Cl.sup.-]-free (N[O.sup.-.sub.3]) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a [Cl.sub.-] channel blocker) decreased it and eliminated the enhancement induced by bumetanide or N[O.sup-.sub.3] solution. ACh and [Ca.sup.2+] dose-response studies demonstrated that N[O.sup-.sub.3] solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of N[O.sup-.sub.3] solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of N[O.sup-.sub.3] solution after ATP depletion induced only a sustained phase. Intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sup.i]) measurements showed that bumetanide and N[O.sup-.sub.3] solution enhanced the ACh-stimulated [[[Ca.sup.2+]].sup.i] increase. Measurements of [[[Cl.sub.-]].sub.i] revealed that ACh decreases [[[C1.sup.-]].sub.i] and that bumetanide and N[O.sup-.sub.3] solution decreased [[[C1.sup.-]].sub.i] and enhanced the ACh-evoked [[[Cl.sub.-]].sub.i] decrease; in contrast, NPPB increased [[[C1.sub.-]].sub.i] and inhibited the [[[Cl.sub.-]].sub.i] decrease induced by ACh, bumetanide, or N[O.sup-.sub.3] solution. These suggest that [[[Cl.sub.-]].sub.i] modulates [[[Ca.sup.2+]].sub.i] increase and ATP-dependent priming. In conclusion, a decrease in [[[C1.sub.-]].sub.i] accelerates ATP-dependent priming and [[[Ca.sup.2+]].sub.i] increase, which enhance [Ca.sup.2+]-regulated exocytosis in ACh-stimulated antral mucous cells. gastric antrum; mucin exocytosis; acetylcholine; intracellular [Cl.sub.-] concentration

Synapsin proteins are implicated in synaptic maintenance and in vesicle supply for exocytosis. Synapsin I has been observed to bind and be regulated by calmodulin. It is now shown that a representative of synapsin II binds calmodulin. Calmodulin binding by synapsins is not confined to mammals and it is concluded that calmodulin binding to synapsins could be a general aspect of synaptic function regulation.

A chimera of the temperature-sensitive glycoprotein of vesicular stomatitis virus and green fluorescent protein (ts-G-GFPct) was used to visualize exocytic transport from the endoplasmic reticulum (ER) to the Golgi complex. Ts-G-GFPct concentrates into COPII-positive structures close to the ER, on shift to permissive temperature, forming an intermediate compartment containing ERGIC-53. This indicates a sequential mode of COPII and COPI action.

The dynamics at the plasma membrane resulting from secretory vesicle docking and fusion and compensatory endocytosis has been difficult to observe in living cells primarily due to limited resolution at the light microscopic level. Using the atomic force microscope, we have been able to image and record changes in plasma membrane structure at ultrahigh resolution after stimulation of secretion from isolated pancreatic acinar cells. 'Pits' measuring 500-2000 nm and containing 3-20 depressions measuring 100-180 nm in diameter were observed only at the apical region of acinar cells. The time course of an increase and decrease in 'depression' size correlated with an increase and decrease of amylase secretion from live acinar cells. Depression dynamics and amylase release were found to be regulated in part by actin. No structural changes were identified at the basolateral region of these cells. Our results suggest depressions to be the fusion pores identified earlier in mast cells by freeze-fracture electron microscopy and by electrophysiological measurements. The atomic force microscope has enabled us to observe plasma membrane dynamics of the exocytic process in living cells in real time.

The release of neurotransmitters through exocytosis pores in secretory vesicles is controlled by pore size and the ion exchange between vesicle matrix and the external electrolyte. Electrically charged secretory substances are stored by electrostatic binding to the vesicle matrix and released by ion exchange. The observed kinetics of serotonin release from secretory granules in beige mast cells are similar to those expected for kinetics limited by ion exchange. The pore undergoes several opening and closing events to release small amounts of neurotransmitter.

The mouse sperm surface galactosyltransferase acts as a receptor for the zona pellucida, acellular coat that surrounds the egg during fertilization. The galactosyltransferase does not help in signal transduction events resulting in acrosomal exocytosis. The mouse zona pelludica consists of ZP1, ZP2 and ZP3 glycoproteins. The ZP3 serves as the adhesion ligand for the sperm that initiates acrosome reaction.

The somatostatin blocks the secretin-induced ductal hypercholeresis and exocytosis by cholangiocytes. The effects of secretin and ductal bile secretion in the hepatocytes of the normal rat show that somatostatin receptor gene SSTR(sub 2) is expressed in cholangiocytes after bile duct ligation (BDL). The secretin regulates the ductal bile secretion in BDL by altering the cAMP-dependent exocytosis. The somatostatin affects the receptors present on liver cholangiocytes.

Threshold sensitivity characterized at the cellular level determines the quantity of secretion in reaction to a suboptimal stimulus for permeabilized and Ca2+ ionophore-incited complete mast cells. The level of exocytosis of separate cells is not complete for a suboptimal stimulus produced by a cell surface-directed ligand.

The urea and water transport pathways are monitored by certain physical processes, which provide information to study the theoretical approach of the process. A chaotic kinetic behavior is hypothesized by two- and three-state kinetic models of the apical membrane permeability activation and inactivation. The presumed controlling of the rate constants by vasopressin influences the activation and inactivation of the kinetics.

One of the primary integral membrane proteins of synaptic vesicles is synaptotagmin, which docks vesicles to the release sites, acts as the doubly ionized calcium sensor for the release process and fuses the protein during exocytosis. The genetic analysis of the synaptotagmin gene in Drosophila reveals the functional properties of this protein. Five lethal alleles of synaptotagmin are discovered by the analysis, and one of them has no detectable protein.

Author(s): Tolga Soykan (corresponding author) [1]; Volker Haucke (corresponding author) [1] Cells communicate with each other by secreting small messenger molecules such as hormones or growth factors, many of which [...]

In this issue, Duran et al. (2010. J. Cell Biol. doi: 10.1083/ jcb.200911154) and Manjithaya et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200911149) use yeast genetics to reveal a role for autophagosome intermediates in the unconventional secretion of an acyl coenzyme A (CoA)--binding protein that lacks an endoplasmic reticulum signal sequence. Medium-chain acyl CoAs are also required and may be important for substrate routing to this pathway. doi/ 10.1083/job.201001121