Demeersseman, L. | bioRxiv Science

bioRxiv2024

Highly adaptable deep-learning platform for automated detection and analysis of vesicle exocytosis

Vesicle exocytosis is a fundamental component of intercellular communication, in all organisms. It has been studied for decades, using various imaging tools. Nevertheless, exocytosis research is still limited by the lack of reliable automated analysis procedures. To address this, we developed the Intelligent Vesicle Exocytosis Analysis Platform (IVEA), a nearly universal solution for analyzing exocytosis acquired with live cell imaging. IVEA is applicable to a wide variety of experimental model systems, microscopes and reporter fluorophores. IVEA combines state-of-the-art deep-learning and computer vision regimes to enable fully automated analysis of large data. IVEA runs as a FIJI plugin and does not require prior training or human intervention. IVEA is 60 times faster than manual analysis and is able to detect rare events often missed by the human eye. Overall, IVEA represents a breakthrough in the analysis of cellular secretory mechanisms and has a transformative potential for the exocytosis imaging field.

bioengineering↗

bioRxiv2024

Pore formation at the lytic synapse triggers the canonical pyroptotic cell death pathway

Prokaryotic pore-forming toxins drive inflammasome activation and pyroptosis through K+-dependent activation of the canonical NLRP3/caspase-1/gasdermin D signaling axis. In this study, we hypothesized that perforin, a eukaryotic pore-forming protein released into the lytic synapse by antigen-specific cytotoxic T lymphocytes (CTLs) upon cognate antigen recognition, mimics the pro-pyroptotic activity of ancestral pore-forming toxins, complementing its role as a conduit for granzymes. Utilizing imaging and molecular approaches, we demonstrate that perforation of target cells upon CTL attack elicits swift K+ efflux followed by NLRP3-dependent activation of proinflammatory caspase-1 and its major substrate, the pyroptotic executioner gasdermin D (GSDMD). Acute target cell death upon CTL attack is gasdermin-dependent and demonstrates morphological and molecular features of pyroptosis, including pyroptotic body formation, cell bloating, plasma membrane rupture, and release of intracellular contents. Perforation of target cells by soluble perforin is sufficient to trigger rapid K+ efflux, caspase-1 activation, and pyroptosis. By contrast, sustained interaction with CTLs unmasks a delayed apoptotic phenotype in the remaining target cells. Interestingly, exposure of target cells to exogenous supramolecular attack particles (SMAPs) recapitulates this apoptotic phenotype, suggesting that soluble perforin and SMAPs play dichotomous roles in target cell death. Our results reveal a novel mechanism for engagement of pyroptotic machinery upon CTL attack, in which perforin itself can autonomously engage programmed cell death (PCD), highlighting the complexity and diversity of the CTL lytic arsenal.

immunology↗