Required minimal protein domain of flower for synaptobrevin2 endocytosis in cytotoxic T cells.

Flower, a highly conserved protein, crucial for endocytosis and cellular fitness, has been implicated in cytotoxic T lymphocyte (CTL) killing efficiency through its role in cytotoxic granule (CG) endocytosis at the immune synapse (IS). This study explores the molecular cues that govern Flower-mediated CG endocytosis by analyzing uptake of Synaptobrevin2, a protein specific to CG in mouse CTL. Using immunogold electron microscopy and total internal fluorescence microscopy, we found that Flower translocates in a stimulus-dependent manner from small vesicles to the IS, thereby ensuring specificity in CG membrane protein recycling.

Lytic granule exocytosis at immune synapses: lessons from neuronal synapses.

Regulated exocytosis is a central mechanism of cellular communication. It is not only the basis for neurotransmission and hormone release, but also plays an important role in the immune system for the release of cytokines and cytotoxic molecules. In cytotoxic T lymphocytes (CTLs), the formation of the immunological synapse is required for the delivery of the cytotoxic substances such as granzymes and perforin, which are stored in lytic granules and released exocytosis.

Study of Effector CD8+ T Cell Interactions with Cortical Neurons in Response to Inflammation in Mouse Brain Slices and Neuronal Cultures.

Cytotoxic CD8+ T cells contribute to neuronal damage in inflammatory and degenerative CNS disorders, such as multiple sclerosis (MS). The mechanism of cortical damage associated with CD8+ T cells is not well understood. We developed in vitro cell culture and ex vivo brain slice co-culture models of brain inflammation to study CD8+ T cell-neuron interactions.

Identification of distinct cytotoxic granules as the origin of supramolecular attack particles in T lymphocytes.

Cytotoxic T lymphocytes (CTL) kill malignant and infected cells through the directed release of cytotoxic proteins into the immunological synapse (IS). The cytotoxic protein granzyme B (GzmB) is released in its soluble form or in supramolecular attack particles (SMAP). We utilize synaptobrevin2-mRFP knock-in mice to isolate fusogenic cytotoxic granules in an unbiased manner and visualize them alone or in degranulating CTLs.

Remodelling of Ca homeostasis is linked to enlarged endoplasmic reticulum in secretory cells.

The endoplasmic reticulum (ER) is extensively remodelled during the development of professional secretory cells to cope with high protein production. Since ER is the principal Ca store in the cell, we characterised the Ca homeostasis in NALM-6 and RPMI 8226 cells, which are commonly used as human pre-B and antibody secreting plasma cell models, respectively. Expression levels of Sec61 translocons and the corresponding Sec61-mediated Ca leak from ER, Ca storage capacity and store-operated Ca entry were significantly enlarged in the secretory RPMI 8226 cell line.

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.

A Structure-Activity Relationship Study of Bimodal BODIPY-Labeled PSMA-Targeting Bioconjugates.

The aim of this study was to identify a high-affinity BODIPY peptidomimetic that targets the prostate-specific membrane antigen (PSMA) as a potential bimodal imaging probe for prostate cancer. For the structure-activity study, several BODIPY (difluoroboron dipyrromethene) derivatives with varying spacers between the BODIPY dye and the PSMA Glu-CO-Lys binding motif were prepared. Corresponding affinities were determined by competitive binding assays in PSMA-positive LNCaP cells.

Studying the biology of cytotoxic T lymphocytes in vivo with a fluorescent granzyme B-mTFP knock-in mouse.

Understanding T cell function in vivo is of key importance for basic and translational immunology alike. To study T cells in vivo, we developed a new knock-in mouse line, which expresses a fusion protein of granzyme B, a key component of cytotoxic granules involved in T cell-mediated target cell-killing, and monomeric teal fluorescent protein from the endogenous locus. Homozygous knock-ins, which are viable and fertile, have cytotoxic T lymphocytes with endogeneously fluorescent cytotoxic granules but wild-type-like killing capacity.

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.

Role of V-ATPase a3-Subunit in Mouse CTL Function.

CTLs release cytotoxic proteins such as granzymes and perforin through fusion of cytotoxic granules (CG) at the target cell interface, the immune synapse, to kill virus-infected and tumorigenic target cells. A characteristic feature of these granules is their acidic pH inside the granule lumen, which is required to process precursors of granzymes and perforin to their mature form. However, the role of acidic pH in CG maturation, transport, and fusion is not understood.

Orai, STIM, and PMCA contribute to reduced calcium signal generation in CD8 T cells of elderly mice.

Ca is a crucial second messenger for proper T cell function. Considering the relevance of Ca signals for T cell functionality it is surprising that no mechanistic insights into T cell Ca signals from elderly individuals are reported. The main Ca entry mechanism in T cells are STIM-activated Orai channels.

Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7.

Cytotoxic T lymphocytes kill infected or malignant cells through the directed release of cytotoxic substances at the site of target cell contact, the immunological synapse. While genetic association studies of genes predisposing to early-onset life-threatening hemophagocytic lymphohistiocytosis has identified components of the plasma membrane fusion machinery, the identity of the vesicular components remain enigmatic. Here, we identify VAMP7 as an essential component of the vesicular fusion machinery of primary, human T cells.

AXER is an ATP/ADP exchanger in the membrane of the endoplasmic reticulum.

To fulfill its role in protein biogenesis, the endoplasmic reticulum (ER) depends on the Hsp70-type molecular chaperone BiP, which requires a constant ATP supply. However, the carrier that catalyzes ATP uptake into the ER was unknown. Here, we report that our screen of gene expression datasets for member(s) of the family of solute carriers that are co-expressed with BiP and are ER membrane proteins identifies SLC35B1 as a potential candidate.

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination.

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment.

Does Erythropoietin Regulate TRPC Channels in Red Blood Cells?

Background: Cation channels play an essential role in red blood cells (RBCs) ion homeostasis. One set of ion channels are the transient receptor potential channels of canonical type (TRPC channels). The abundance of these channels in primary erythroblasts, erythroid cell lines and RBCs was associated with an increase in intracellular Ca2+ upon stimulation with Erythropoietin (Epo).

Preparing the lethal hit: interplay between exo- and endocytic pathways in cytotoxic T lymphocytes.

Cytotoxic T lymphocytes patrol our body in search for infected cells which they kill through the release of cytotoxic substances contained in cytotoxic granules. The fusion of cytotoxic granules occurs at a specially formed contact site, the immunological synapse, and is tightly controlled to ensure specificity. In this review, we discuss the contribution of two intracellular compartments, endosomes and cytotoxic granules, to the formation, function and disassembly of the immunological synapse.

Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes.

CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner.

The Disease Protein Tulp1 Is Essential for Periactive Zone Endocytosis in Photoreceptor Ribbon Synapses.

Mutations in the Tulp1 gene cause severe, early-onset retinitis pigmentosa (RP14) in humans. In the retina, Tulp1 is mainly expressed in photoreceptors that use ribbon synapses to communicate with the inner retina. In the present study, we demonstrate that Tulp1 is highly enriched in the periactive zone of photoreceptor presynaptic terminals where Tulp1 colocalizes with major endocytic proteins close to the synaptic ribbon.

Molecular Plasticity of Male and Female Murine Gonadotropes Revealed by mRNA Sequencing.

Gonadotropes in the anterior pituitary gland are of particular importance within the hypothalamic-pituitary-gonadal axis because they provide a means of communication and thus a functional link between the brain and the gonads. Recent results indicate that female gonadotropes may be organized in the form of a network that shows plasticity and adapts to the altered endocrine conditions of different physiological states. However, little is known about functional changes on the molecular level within gonadotropes during these different conditions.

Podoplanin discriminates distinct stromal cell populations and a novel progenitor subset in the liver.

Podoplanin/gp38(+) stromal cells present in lymphoid organs play a central role in the formation and reorganization of the extracellular matrix and in the functional regulation of immune responses. Gp38(+) cells are present during embryogenesis and in human livers of primary biliary cirrhosis. Since little is known about their function, we studied gp38(+) cells during chronic liver inflammation in models of biliary and parenchymal liver fibrosis and steatohepatitis.

Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells.

Pannexins (Panx) are proteins with a similar membrane topology to connexins, the integral membrane protein of gap junctions. Panx1 channels are generally of major importance in a large number of system and cellular processes and their function has been thoroughly characterized. In contrast, little is known about channel structure and subcellular distribution.

Secretion and immunogenicity of the meningioma-associated antigen TXNDC16.

In a previous study, we identified thioredoxin domain containing 16 (TXNDC16) as a meningioma-associated Ag by protein macroarray screening. Serological screening detected autoantibodies against TXNDC16 exclusively in meningioma patients' sera and not in sera of healthy controls. TXNDC16 was previously found to be an endoplasmic reticulum (ER)-luminal glycoprotein.

In the crosshairs: investigating lytic granules by high-resolution microscopy and electrophysiology.

Cytotoxic T lymphocytes (CTLs) form an integral part of the adaptive immune system. Their main function is to eliminate bacteria- and virus-infected target cells by releasing perforin and granzymes (the lethal hit) contained within lytic granules (LGs), at the CTL-target-cell interface [the immunological synapse (IS)]. The formation of the IS as well as the final events at the IS leading to target-cell death are both highly complex and dynamic processes.

Syntaxin11 serves as a t-SNARE for the fusion of lytic granules in human cytotoxic T lymphocytes.

CTLs kill target cells via fusion of lytic granules (LGs) at the immunological synapse (IS). Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) function as executors of exocytosis. The importance of SNAREs in CTL function is evident in the form of familial hemophagocytic lymphohistiocytosis type 4 that is caused by mutations in Syntaxin11 (Stx11), a Qa-SNARE protein.