Investigation of Cytotoxic T Lymphocyte Function during Allorejection in the Anterior Chamber of the Eye

Cytotoxic T lymphocytes (CTL) are an essential part of our immune system by killing infected and malignant cells. To fully understand this process, it is necessary to study CTL function in the physiological setting of a living organism to account for their interplay with other immune cells like CD4+ T helper cells and macrophages. The anterior chamber of the eye (ACE), originally developed for diabetes research, is ideally suited for non-invasive and longitudinal in vivo imaging. We take advantage of the ACE window to observe immune responses, particularly allorejection of islets of Langerhans cells by CTLs. We follow the onset of the rejection after vascularization on islets until the end of the rejection process for about a month by repetitive two-photon microscopy. We find that CTLs show reduced migration on allogeneic islets in vivo compared to in vitro data, indicating CTL activation. Interestingly, the temporal infiltration pattern of T cells during rejection is precisely regulated, showing enrichment of CD4+ T helper cells on the islets before arrival of CD8+ CTLs. The adaptation of the ACE to immune responses enables the examination of the mechanism and regulation of CTL-mediated killing in vivo and to further investigate the killing in gene-deficient mice that resemble severe human immune diseases

Interactions with membrane lipids and SNARE complex underlie dual roles for calcium-dependent activator protein for secretion (CAPS) in large dense-core vesicles (LDCVS) priming

CAPS (Calcium-dependent activator protein for secretion), was discovered as a soluble factor required for catecholamine secretion from PC12 cells. CAPS facilitates release of dense-core vesicles and release of neurotransmitters. In vertebrates CAPS is required for priming of synaptic vesicles and dense-core vesicles in neurons and in catecholamine release from the adrenal medulla. The pleckstrin homology domain of CAPS has been shown to mediate an interaction with the plasma membrane. An interaction with SNARE proteins and assembled SNARE complexes requires the Munc13 homology domain. The interaction with SNARE components facilitates SNARE complex formation, the molecular basis of the priming reaction, while interactions with the phosphoinositol 4,5 bisphosphate (PI(4,5)P2) rich areas of the plasma membrane localize CAPS at the release site but may also promote the fusion of primed vesicles. CAPS splice variants lacking the Munc13 homology domain but with an intact pleckstrin homology domain promote priming to the readily releasable pool while CAPS splice variants with a deletion in the pleckstrin homology domain promote catecholamine release, albeit at slower rates. These results confirm a dual action of CAPS dependent on the Munc13 homology- and the pleckstrin homology domains, respectively.Sociedad Argentina de Fisiologí

Various Stages of Immune Synapse Formation Are Differently Dependent on the Strength of the TCR Stimulus

Cytotoxic T lymphocytes (CTL) are key players of the adaptive immune system that target tumors and infected cells. A central step to that is the formation of a cell–cell contact zone between the CTL and its target called an immune synapse (IS). Here, we investigate the influence of the initial T cell receptor (TCR) trigger of a cytolytic IS on the distinct steps leading to cytotoxic granule (CG) exocytosis. We stimulated primary CTLs from mouse using lipid bilayers with varying anti-CD3 but constant ICAM concentrations. We fluorescently labeled molecular markers of distinct IS zones such as actin, CD3, granzyme B, and Synaptobrevin2 in CTLs and imaged cytolytic IS formation by total internal reflection fluorescence microscopy (TIRFM). We found that an intermediate anti-CD3 concentration of 10 µg/mL induces the fastest adhesion of CTLs to the bilayers and results in maximal CG fusion efficiency. The latency of actin ring formation, dwell time, and maximum surface area at the IS exhibit different dependencies on the stimulatory anti-CD3 concentrations. The number and surface area of CD3 clusters at the IS seem to show a different dependency to the TCR trigger when compared to their dwell time. Finally, the mode of full CG exocytosis appears to be independent of the TCR trigger

RGS2 Determines Short-Term Synaptic Plasticity in Hippocampal Neurons by Regulating Gi/o- Mediated Inhibition of Presynaptic Ca2+ Channels

SummaryRGS2, one of the small members of the regulator of G protein signaling (RGS) family, is highly expressed in brain and regulates Gi/o as well as Gq-coupled receptor pathways. RGS2 modulates anxiety, aggression, and blood pressure in mice, suggesting that RGS2 regulates synaptic circuits underlying animal physiology and behavior. How RGS2 in brain influences synaptic activity is unknown. We therefore analyzed the synaptic function of RGS2 in hippocampal neurons by comparing electrophysiological recordings from RGS2 knockout and wild-type mice. Our study provides a general mechanism of the action of the RGS family containing RGS2 by demonstrating that RGS2 increases synaptic vesicle release by downregulating the Gi/o-mediated presynaptic Ca2+ channel inhibition and therefore provides an explanation of how regulation of RGS2 expression can modulate the function of neuronal circuits underlying behavior

CAPS facilitates filling of the rapidly releasable pool of large dense-core vesicles

Calcium-activator protein for secretion (CAPS) is a cytosolic protein that associates with large dense-core vesicles and is involved in their secretion. Mammals express two CAPS isoforms, which share a similar domain structure including a Munc13 homology domain that is believed to be involved in the priming of secretory vesicles. A variety of studies designed to perturb CAPS function indicate that CAPS is involved in the secretion of large dense-core vesicles, but where in the secretory pathway CAPS acts is still under debate. Mice in which one allele of the CAPS-1 gene is deleted exhibit a deficit in catecholamine secretion from chromaffin cells. We have examined catecholamine secretion from chromaffin cells in which both CAPS genes were deleted and show that the deletion of both CAPS isoforms causes a strong reduction in the pool of rapidly releasable chromaffin granules and of sustained release during ongoing stimulation. We conclude that CAPS is required for the adequate refilling and/or maintenance of a rapidly releasable granule pool

P38α-MAPK phosphorylates Snapin and reduces Snapin-mediated BACE1 transportation in APP-transgenic mice

Amyloid β peptide (Aβ) is the major pathogenic molecule in Alzheimer's disease (AD). BACE1 enzyme is essential for the generation of Aβ. Deficiency of p38α-MAPK in neurons increases lysosomal degradation of BACE1 and decreases Aβ deposition in the brain of APP-transgenic mice. However, the mechanisms mediating effects of p38α-MAPK are largely unknown. In this study, we used APP-transgenic mice and cultured neurons and observed that deletion of p38α-MAPK specifically in neurons decreased phosphorylation of Snapin at serine, increased retrograde transportation of BACE1 in axons and reduced BACE1 at synaptic terminals, which suggests that p38α-MAPK deficiency promotes axonal transportation of BACE1 from its predominant locations, axonal terminals, to lysosomes in the cell body. In vitro kinase assay revealed that p38α-MAPK directly phosphorylates Snapin. By further performing mass spectrometry analysis and site-directed mutagenic experiments in SH-SY5Y cell lines, we identified serine residue 112 as a p38α-MAPK-phosphorylating site on Snapin. Replacement of serine 112 with alanine did abolish p38α-MAPK knockdown-induced reduction of BACE1 activity and protein level, and transportation to lysosomes in SH-SY5Y cells. Taken together, our study suggests that activation of p38α-MAPK phosphorylates Snapin and inhibits the retrograde transportation of BACE1 in axons, which might exaggerate amyloid pathology in AD brain

Tomosyn Expression Pattern in the Mouse Hippocampus Suggests Both Presynaptic and Postsynaptic Functions

The protein tomosyn decreases synaptic transmission and release probability of vesicles, and is essential for modulating synaptic transmission in neurons. In this study, we provide a detailed description of the expression and localization patterns of tomosyn1 and tomosyn2 in the subareas of the mouse hippocampus. Using confocal and two-photon high-resolution microscopy we demonstrate that tomosyn colocalizes with several pre- and postsynaptic markers and is found mainly in glutamatergic synapses. Specifically, we show that tomosyn1 is differentially distributed in the mouse hippocampus and concentrated mainly in the hilus and mossy fibers. Surprisingly, we found that tomosyn2 is expressed in the subiculum, CA1 and CA2 pyramidal cell bodies, dendrites and spines, and colocalizes with PSD95, suggesting a postsynaptic role. These results suggest that in addition to the well-characterized presynaptic function of tomosyn in neurotransmitter release, tomosyn2 might have a postsynaptic function, and place tomosyn as a more general regulator of synaptic transmission and plasticity

CAPS1 Regulates Catecholamine Loading of Large Dense-Core Vesicles

SummaryCAPS1 is thought to play an essential role in mediating exocytosis from large dense-core vesicles (LDCVs). We generated CAPS1-deficient (KO) mice and studied exocytosis in a model system for Ca2+-dependent LDCV secretion, the adrenal chromaffin cell. Adult heterozygous CAPS1 KO cells display a gene dosage-dependent decrease of CAPS1 expression and a concomitant reduction in the number of docked vesicles and secretion. Embryonic homozygous CAPS1 KO cells show a strong reduction in the frequency of amperometrically detectable release events of transmitter-filled vesicles, while the total number of fusing vesicles, as judged by capacitance recordings or total internal reflection microscopy, remains unchanged. We conclude that CAPS1 is required for an essential step in the uptake or storage of catecholamines in LDCVs

Penggambaran superhero pada tokoh Deadpool dalam film Deadpool

Deadpool merupakan film superhero yang diproduksi oleh Marvel Studio pada tahun 2016. Film ini menceritakan sosok superhero yang memiliki sisi lain dari superhero pada umumnya. Penelitian ini bertujuan untuk melihat bagaimana tanda-tanda penggambaran superhero pada tokoh Deadpool dalam film Deadpool. Superhero merupakan sosok yang memiliki kekuatan diluar nalar manusia dan mereka selalu menutupi identitasnya. Metode analisis semiotika Charles Sanders Pierce digunakan untuk meneliti tanda penggambaran pada tokoh Deadpool dalam film Deadpool. Melalui tanda-tanda yang muncul dalam film Deadpool, peneliti menemukan bahwa menjadi superhero tidak harus menjadi sosok yang sempurna baik fisik maupun sifatnya. Selain itu film ini mempertegas bahwa menjadi superhero hanya membutuhkan kemauan yang kuat, jiwa kepahlawanan yang besar dan usaha yang maksimal