The role of Flower splice variants in regulating endocytosis in primary cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTLs) are important immune defense fighters, attacking intracellular pathogens such as viruses and bacteria, and are major players in tumour surveillance. The immunological synapse (IS) between T cells and antigen presenting cells is the focal point for exocytosis of cytotoxic granule contents leading to target cell apoptosis and/or lysis. Exo- and endocytosis are among the most fundamental pathways in cells. Both of these pathways are highly interdependent, as several proteins contribute to the dynamic function. The transmembrane protein Flower (Fwe) is associated with synaptic vesicle endocytosis in neurons and is also known for its important role in regulating cytotoxic granule (CG) endocytosis in primary cytotoxic T lymphocytes (CTLs). The major aim of this study is to characterize the function of Flower splice variants in the endocytosis of CG material and to subsequently decipher the mechanism of Flower ҆s role in endocytosis. Flower is expressed in primary mouse CD8+ T cells and is upregulated during differentiation from naïve to effector cells. RT-PCR shows the expression of four mouse Flower isoforms (FweA, B, D and E) in brain and activated CTLs. Real-time killing assays with different effector: target cell ratios showed a clear defect in killing efficacy of Flower deficient CTLs in comparison to wild-type cells. Fwe deficient CTLs showed accumulation of the CG marker protein Synaptobrevin2 (Syb2) in the plasma membrane at the IS indicating that it is not endocytosed. Since Transferrin receptor1 (TfR1) internalization into the cells was not affected, it appears that endocytosis of a CG-independent protein was not completely blocked. Only reintroduction of FweA, the longest isoform, was able to fully rescue Syb2 endocytosis while introduction of FweB and E showed partial rescue. Expression of FweD did not rescue Syb2 endocytosis and resulted in behavior similar to Fwe KO cells. The incomplete rescue by FweB and E was likely caused by differences in the C-terminus region. Comparison of the rescue phenotype induced by drosophila and human Flower isoforms gave indicates that the C-terminus (140-171 aa) and conserved exon3 (81-99 aa) regions are the major regions involved in aiding Syb2 endocytosis. Various mutated constructs involving the YXXɸ motif region YRWL and YARI at the N- and C-termini and mutation of the highly conserved tyrosine residue (Y104A) in the exon3 region showed no rescue of endocytosis which might indicate the importance of the Flower isoforms in various functional and downstream pathways of endocytosis. Flower topology studies using Halo tag-Atto488 staining show that the N- and C-termini of Flower are cytosolic. CD3/CD28 bead activation triggers the movement of Flower to the plasma membrane where it is mainly localized at the IS. A vesicular localization of the Flower constructs which rescue endocytosis was seen specifically at the IS whereas the Flower constructs which did not rescue endocytosis were localized to the endoplasmic reticulum (ER). Inability to traffic to the plasma membrane likely explains the inability of some isoforms to support endocytosis. Thus, the results of this study have allowed us to characterize the role of the naturally truncated splice variants in regulating CG endocytosis in primary murine CTLs.Zytotoxische T-Lymphozyten (CTLs) sind wichtige zelluläre Komponenten der Abwehr von intrazellulären Krankheitserregern wie Viren und Bakterien und spielen eine wichtige Rolle bei der Tumorüberwachung. Die immunologische Synapse (IS) zwischen T-Zellen und Antigen-präsentierenden Zellen ist der Ort der Exozytose von zytotoxischen Granulainhalten, die zur Apoptose und/oder Lyse der Zielzellen führt. Exo- und Endozytose sind einer der grundlegendsten Prozesse in allen Zellen. Beide Prozesse sind stark voneinander abhängig, da mehrere Proteine zusammenspielen und zur dynamischen Funktion in verschiedenen Zelltypen beitragen. Es wurde gezeigt, dass das Transmembranprotein Flower (Fwe) mit der synaptischen Vesikelendozytose in Neuronen assoziiert ist und ebenfalls eine wichtige Rolle bei der Regulierung der zytotoxischen Granula (CG)-Endozytose in primären zytotoxischen T-Lymphozyten (CTLs) hat. Das Hauptziel der vorliegenden Arbeit ist es, die Funktion von Flower-Spleißvarianten zu charakterisieren und anschließend einen möglichen Mechanismus zu entschlüsseln, wie Flower die Endozytose cytotoxischer Granulabestandteile unterstützt. Flower wird in primären Maus-CD8+-T-Zellen exprimiert und wird während der Reifung zu Effektorzellen hochreguliert. RT-PCR zeigt die Expression von vier Maus-Flower-Isoformen (FweA, B, D und E) im Gehirn und aktivierten CTLs. Killing Assays mit unterschiedlichem Effektor:Zielzellen Verhältnis zeigten einen klaren Defekt in der Abtötungseffizienz von Flower-defizienten CTLs im Vergleich zu Wildtypzellen. Außerdem zeigten Fwe-defiziente CTLs eine Akkumulation des CG-Markers Synaptobrevin2 (Syb2) an der IS, was für einen Endozytosedefekt spricht. Die Internalisierung des Transferrin-Rezeptors1 (TfR1) war jedoch nicht beeinträchtigt. Die Wiedereinführung von Fwe-Isoformen zeigte, dass nur die längste Isoform FweA den Syb2-Block überwinden konnte, während FweB und E einen teilweisen Effekt zeigten. FweD konnte die Syb2-Akkumulation nicht verhindern. Überexprimierende Zellen verhielten sich ähnlich wie Fwe KO-Zellen. Die nur teilweise Wiederherstellung der Endozytose in Fwe-KO-CTLs durch die Maus-FweB- und -E-Isoformen im Vergleich zur A-Variante ist wahrscheinlich auf Unterschiede in der C-Terminus-Region zurückzuführen. Der Vergleich des Rescue-Phänotyps von Drosophila und humanen Fwe Isoformen gab uns den Hinweis, dass der C-Terminus (140-171 aa) und die konservierten Exon3-Regionen (81-99 aa) die Hauptregionen sind, die an der Unterstützung der Syb2-Endocytose beteiligt sind. Verschiedene Mutationen der YXXɸ-Motivregionen YRWL und YARI an den N- und C-Termini und Mutationen des hochkonservierten Tyrosinrests (Y104A) in der Exon3-Region zeigten keine Wiederherstellung der Endozytose, was auf verschiedene Funktionen der Flower-Isoformen für verschiedene Schritte der Endozytose hinweisen könnte. Studien zur Flower-Topologie mit Halo-Tag-Atto488-Färbung zeigen, dass die N- und C-Termini von Flower zytosolisch sind. CD3/CD28-Bead-Aktivierung löst die Translokalisation von Flower an die Plasmamembran und hier hauptsächlich zur IS aus. Eine vesikuläre Lokalisation der Endozytose-wiederherstellenden Flower-Konstrukte wurde speziell an der IS beobachtet, während die erfolglosen Flower-Konstrukte eine Lokalisierung am endoplasmatischen Retikulum (ER) zeigten, was die unterschiedlichen funktionellen Aspekte der verschiedenen Isoformen betont. Die Ergebnisse dieser Studie können helfen, die Rolle der Spleißvarianten bei der Regulierung der CG-Endocytose in primären murinen CTLs zu verstehen

NCS-1 Deficiency Is Associated With Obesity and Diabetes Type 2 in Mice

Neuronal calcium sensor-1 (NCS-1) knockout (KO) in mice (NCS-1−/− mice) evokes behavioral phenotypes ranging from learning deficits to avolition and depressive-like behaviors. Here, we showed that with the onset of adulthood NCS-1−/− mice gain considerable weight. Adult NCS-1−/− mice are obese, especially when fed a high-fat diet (HFD), are hyperglycemic and hyperinsulinemic and thus develop a diabetes type 2 phenotype. In comparison to wild type (WT) NCS-1−/− mice display a significant increase in adipose tissue mass. NCS-1−/− adipocytes produce insufficient serum concentrations of resistin and adiponectin. In contrast to WT littermates, adipocytes of NCS-1−/− mice are incapable of up-regulating insulin receptor (IR) concentration in response to HFD. Thus, HFD-fed NCS-1−/− mice exhibit in comparison to WT littermates a significantly reduced IR expression, which may explain the pronounced insulin resistance observed especially with HFD-fed NCS-1−/− mice. We observed a direct correlation between NCS-1 and IR concentrations in the adipocyte membrane and that NCS-1 can be co-immunoprecipitated with IR indicating a direct interplay between NCS-1 and IR. We propose that NCS-1 plays an important role in adipocyte function and that NCS-1 deficiency gives rise to obesity and diabetes type 2 in adult mice. Given the association of altered NCS-1 expression with behaviorial abnormalities, NCS-1−/− mice may offer an interesting perspective for studying in a mouse model a potential genetic link between some psychiatric disorders and the risk of being obese

Faster cytotoxicity with age : Increased perforin and granzyme levels in cytotoxic CD8+ T cells boost cancer cell elimination

A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8+ T cells responses in the elderly has come back into focus since the COVID-19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8+ T cells in aging. We focused on the different subpopulations and time-resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8+ T cells from elderly mice are much faster than those of CD8+ T cells from adult mice. This is true not only in the total CD8+ T cell population but also for their effector (TEM) and central memory (TCM) T cell subpopulations. TIRF experiments reveal that CD8+ T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8+ T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell-intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked

Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Melanosomes are a type of lysosome-related organelle that is commonly defective in Hermansky-Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC-1, -2, -3, or AP-1, -3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A-depletion phenotype resembles Rab38/ 32-inactivated or BLOC-3-deficient melanocytes, suggesting that Rab9A works in line with BLOC-3 and Rab38/ 32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/ 32 or its effector VARP, or BLOC-3-deficiency in melanocytes decreased the length of STX13-positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co-regulatory GTPases control STX13-mediated cargo delivery to maturing melanosomes

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. We identified two classes of fusion-competent granules, single core granules (SCG) and multi core granules (MCG), with different diameter, morphology and protein composition. Functional analyses demonstrate that both classes of granules fuse with the plasma membrane at the IS. SCG fusion releases soluble GzmB. MCGs can be labelled with the SMAP marker thrombospondin-1 and their fusion releases intact SMAPs. We propose that CTLs use SCG fusion to fill the synaptic cleft with active cytotoxic proteins instantly and parallel MCG fusion to deliver latent SMAPs for delayed killing of refractory targets

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. We identified two classes of fusion-competent granules, single core granules (SCG) and multi core granules (MCG), with different diameter, morphology and protein composition. Functional analyses demonstrate that both classes of granules fuse with the plasma membrane at the IS. SCG fusion releases soluble GzmB. MCGs can be labelled with the SMAP marker thrombospondin-1 and their fusion releases intact SMAPs. We propose that CTLs use SCG fusion to fill the synaptic cleft with active cytotoxic proteins instantly and parallel MCG fusion to deliver latent SMAPs for delayed killing of refractory targets