Early activation of <scp>CD</scp>95 is limited and localized to the cytotoxic synapse

Sánchez, M. Florencia; Murad, Fabronia; Balta, Gülce S. Gülcüler; Martín-Villalba, Ana; García‐Sáez, Ana J.; Carrer, Dolores C.

doi:10.1111/febs.14518

Cited by 4 publications

(4 citation statements)

References 77 publications

(104 reference statements)

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“…Nano-and microlithographic approaches have provided cell-compatible scaffolds to investigate confined ligand-receptor interactions (Manz & Groves, 2010;Nair et al, 2011;Torres et al, 2008). Various techniques, ranging from photolithography to polymer lift-off and microcontact printing (µCP), have yielded information on how topology and mobility of the stimulus regulate cell responses (Bernard et al, 2000;Doh & Irvine, 2006;Mossman et al, 2005;Sánchez et al, 2018;Traub et al, 2016). So far, these methods utilize receptor ligands or antibodies to induce a rather static cluster formation.…”

Section: Introductionmentioning

confidence: 99%

“…Nano- and microlithographic approaches have provided cell-compatible scaffolds to investigate confined ligand-receptor interactions. Various techniques, ranging from photolithography (Chen et al ., 2021; Scheideler et al ., 2020; Traub et al ., 2016) to electron-beam lithography (Cai et al ., 2018; Nassereddine et al ., 2021) and microcontact printing (μCP) (Lindner et al ., 2018; Sánchez et al ., 2018), have yielded information on how topology and mobility of the stimulus regulate cellular outcomes. Recently, optogenetics and optochemistry have provided the possibility of targeting receptor oligomerization with high spatiotemporal control (Bardhan & Deiters, 2019; Goglia & Toettcher, 2019; Taslimi et al ., 2014).…”

Section: Introductionmentioning

confidence: 99%

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Dynamic in situ confinement triggers ligand-free neuropeptide receptor signaling

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Sánchez

Dietz

et al. 2021

Preprint

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Membrane receptors are central to cell-cell communication. Receptor clustering at the plasma membrane modulates physiological responses, and microscale receptor organization is critical for downstream signaling. Spatially restricted cluster formation of the neuropeptide Y2 hormone receptor (Y2R) was observed in vivo; however, the relevance of this confinement is not fully understood. Here, we controlled Y2R clustering in situ by a multivalent chelator nanotool in prestructured matrices. Fast Y2R enrichment in microscale arrays triggered a ligand-independent downstream signaling determined by an increase in cytosolic calcium, cell spreading and migration. We reveal that ligand-independent signaling by confinement differs from ligand-induced activation in the recruitment of arrestin-3 as downstream effector, which was recruited to confined regions only in presence of the ligand. The employed multivalent nanotool facilitated a dynamic receptor enrichment with high exchange in the confined regions, comparable to microscale condensates. This concept enables in situ organization of membrane receptors and the exploration of ligand-independent receptor signaling.Abstract Figure

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Dynamic in situ confinement triggers ligand-free neuropeptide receptor signaling

Tampé

Sánchez

Dietz

et al. 2021

Preprint

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“…Nano- and microlithographic approaches have provided cell-compatible scaffolds to investigate confined ligand–receptor interactions. Various techniques, ranging from photolithography − to electron-beam lithography , and microcontact printing (μCP), , have yielded information on how topology and mobility of the stimulus regulate cellular outcomes. One of the main drawbacks of these systems is that the cells are in contact with the functionalized matrices for minutes/hours before the response is evaluated, thus missing the early signaling events upon cluster formation.…”

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confidence: 99%

Dynamic in Situ Confinement Triggers Ligand-Free Neuropeptide Receptor Signaling

et al. 2022

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Membrane receptor clustering is fundamental to cell–cell communication; however, the physiological function of receptor clustering in cell signaling remains enigmatic. Here, we developed a dynamic platform to induce cluster formation of neuropeptide Y2 hormone receptors (Y2R) in situ by a chelator nanotool. The multivalent interaction enabled a dynamic exchange of histidine-tagged Y2R within the clusters. Fast Y2R enrichment in clustered areas triggered ligand-independent signaling as determined by an increase in cytosolic calcium and cell migration. Notably, the calcium and motility response to ligand-induced activation was amplified in preclustered cells, suggesting a key role of receptor clustering in sensitizing the dose response to lower ligand concentrations. Ligand-independent versus ligand-induced signaling differed in the binding of arrestin-3 as a downstream effector, which was recruited to the clusters only in the presence of the ligand. This approach allows in situ receptor clustering, raising the possibility to explore different receptor activation modalities.

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“…It could be shown that secretion of two to four cytotoxic granula of NK cells could lead to the induction of target cell death [125]. Both mechanisms, are taking place at the immunological synapse [126,127]. Further, NK cells are known to be serial killer.…”

Section: Natural Killer Cells As Cellular Immunotherapymentioning

confidence: 99%

Molecular dissection of BV6 induced sensitization of rhabdomyosarcoma spheroids towards NK cell attack

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Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in early childhood. Despite recent advances in the treatment regimes of rhabdomyosarcoma, the 5-year survival is still alarmingly low for the more aggressive metastasizing alveolar rhabdomyosarcoma subtype. Novel treatment strategies are needed in order to increase the overall survival rate. Hallmarks of cancer include evade cell death induction and evade immune system surveillance. This is mediated in part by up-regulation of inhibitor of apoptosis (IAP) proteins. With the development of Smac mimetic compounds mimicking the endogenous IAP antagonist Smac, this tumor evasion mechanism became exploitable. In this PhD thesis, a combinatory approach for a putative treatment option of RMS will be presented. Here, the Smac mimetic compound BV6 will be used as a pre-treatment of RMS cells. This leads to a sensitizing effect within the tumor cells, increasing the killing efficacy of natural killer (NK) cells. Subtoxic concentrations of BV6 were chosen to sensitize RMS cells. To remodel the solid tumor characteristics of RMS, a multicellular RMS tumor spheroid culture model was used. In both tumor spheroids and conventional monolayer cell culture BV6 induced the degradation of IAP proteins (cIAP1, cIAP2, in spheroids XIAP). Further, BV6 led to the activation of both, the canonical and non-canonical NF-κB signaling pathways. This was demonstrated by an increased IκBα and p65 phosphorylation, and nuclear translocation of p-p65, indicative for an active canonical NF-κB signaling. On the other side, cIAP degradation led to the stabilization and accumulation of NIK and downstream partial degradation of p100 to p52 and its nuclear translocation, indicating non-canonical NF-κB signaling pathway activity. A bulk RNA sequencing approach of BV6 treated RH30 cells validated the NF-κB signaling involvement and identified 182 differentially expressed genes. Among the interesting target genes are NFKBIA (IκBα),BIRC3 (cIAP2), NFKB2 (p100), CCL5 and SSTR2. SSTR2 was thoroughly validated as being up-regulated on a transcriptional and on protein level. Here, SSTR2A, one of the two alternative splicing variants, is up-regulated and opens a hypothetical targeted treatment strategy, as SSTR2 expression is not associated with RMS, but rather described with neuroendocrine tumor entities. In addition, CCL5 was thoroughly validated as a BV6 induced target. Again, the up-regulated mRNA transcription was validated by an increased translation and by increased secretion of CCL5. As CCL5 being associated as pro-migratory and activating of NK cells, CRISPR/Cas9 mediated CCL5 knock-out studies were performed to evaluate the influence of CCL5 within a BV6 pre-treatment and NK cell co-cultivation setting. It was shown that CCL5 knock-out does not rescue BV6 pre-treated RMS spheroids from NK cell attack and killing. The previous mentioned transcriptional activity by BV6 stimulation was NIK mediated as knock-down of NIK reduced the mRNA transcription of several interesting genes. However, NIK mediated down-stream signaling had no influence on the BV6 induced sensitizing effect towards NK cell mediated attack. A NIK knock-down had no rescue effect upon BV6 pre-treatment and NK cell co-treatment. As cIAP proteins are present in receptor bound complexes, e.g. complex I at the TNF receptor 1 (TNFR1), a putative involvement of death receptors in general was evaluated. Indeed, BV6 treatment of RMS cells could increase the surface presentation of DR5, a death receptor ligating TRAIL. Functionally, co-treatment of BV6 with TRAIL led to an additive cell death inducting effect. However, within the NK cell co-cultivation setting, addition of a neutralizing TRAIL anitbody could not rescue BV6 pre-treated RMS spheroids from NK cell killing. A similar effect was observed when neutralizing TNFα by adding Enbrel during the NK cell co-cultivation. BV6 sensitization of RMS spheroids seems to be independent of death receptors. In addition to activating NF-κB, BV6 as a Smac mimetic is supposed to be able to release caspases bound by IAP proteins. Indeed, BV6 pre-treatment of RMS spheroids and co-cultivation with NK cells could cleave and thereby activate the executioner caspase-3. Further, treatment with a pan-caspase inhibitor, zVAD.fmk, could reduce the BV6 mediated sensitizing effect towards NK cell attack in RD spheroids. Taken together, BV6 does induce a thoroughly validated NF-κB signaling pathway, leading to a NIK mediated transcriptional signature change. However, the NF-κB activation might not be responsible for the observed sensitization. Further, BV6 in combination with NK cells led to a seemingly death receptor independent, caspase dependent cell death induction of RMS spheroids. Although the mechanism remains partially con-cealed, a therapeutic benefit by combining a cell death sensitizing compound, i.e. BV6, with cytotoxic lymphocytes is evident.

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Early activation of CD95 is limited and localized to the cytotoxic synapse

Cited by 4 publications

References 77 publications

Dynamic in situ confinement triggers ligand-free neuropeptide receptor signaling

Dynamic in situ confinement triggers ligand-free neuropeptide receptor signaling

Dynamic in Situ Confinement Triggers Ligand-Free Neuropeptide Receptor Signaling

Molecular dissection of BV6 induced sensitization of rhabdomyosarcoma spheroids towards NK cell attack

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