CASPAM: A Triple-Modality Biosensor for Multiplexed Imaging of Caspase Network Activity

Habif, Martín; Corbat, Agustín Andrés; Silberberg, Mauro; Grecco, Hernán E.

doi:10.1021/acssensors.1c00554

Cited by 6 publications

(5 citation statements)

References 41 publications

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“…Moreover, we also tested Spz cleavage depending on pro-Psh and H 2 O 2 treatment, by co-overexpressing Spz WT or Spz uc and pro-Psh using a single plasmid with the viral P2A sequence between the Spz WT or Spz uc and pro-Psh open reading frames (ORFs) [ 51 , 52 ] (Figs 5A and S7D ). Cleavage of Spz WT occurred depending on H 2 O 2 treatment.…”

Section: Resultsmentioning

confidence: 99%

“…For pMT-Spz-3xFLAG-P2A-pro-Psh WT and pMT-Spz uc -3xFLAG-P2A-pro-Psh WT , the coding sequences of Spz-3xFLAG or Spz uc -3xFLAG were PCR-amplified from pMT-Spz-3xFLAG or pMT-Spz uc -3xFLAG plasmids, respectively, with GGGSGGG linker and P2A sequences at the 3’ terminus [ 51 , 52 ]. The coding sequence of pro-Psh WT was PCR-amplified from pMT-pro-Psh WT .…”

Section: Methodsmentioning

confidence: 99%

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Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies

et al. 2023

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The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spätzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, we revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, we found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE/MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies

et al. 2023

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“…Sample preparation and image acquisition: samples correspond to HeLa cells transfected with CASPAM, a cytosolic biosensor described in Habif et al 2021. Imaging was performed by means of a custom built setup as described in Squire et al 2004 andVilar et al 2009.…”

Section: Methodsmentioning

confidence: 99%

Robust and unbiased estimation of the background distribution for automated quantitative imaging

Silberberg

Grecco

2021

Preprint

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Quantitative analysis of high-throughput microscopy images requires robust automated algorithms. Background estimation is usually the first step and has an impact on all subsequent analysis, in particular for foreground detection and calculation of ratiometric quantities. Most methods recover only a single background value, such as the median. Those that aim to retrieve a background distribution by dividing the intensity histogram yield a biased estimation in images in non-trivial cases. In this work, we present the first method to recover an unbiased estimation of the background distribution directly from an image and without any additional input. Through a robust statistical test, our method leverages the lack of local spatial correlation in background pixels to select a subset of pixels that accurately represent the background distribution. This method is both fast and simple to implement, as it only uses standard mathematical operations and an averaging filter. Additionally, the only parameter, the size of the averaging filter, does not require fine tuning. The obtained background distribution can be used to test for foreground membership of individual pixels, or to estimate confidence intervals in derived quantities. We expect that the concepts described in this work can help to develop a novel family of robust segmentation methods.

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“…The most straightforward solution for multiplexing is to employ spectral distinct biosensors that can be easily distinguished based on their fluorescence properties ( Figure 2a ). Habif et al [ 37 ] reported a triple-modality biosensor system, CASPAM, which expresses three differently colored FP-based biosensors in equimolar concentrations. CASPAM enables simultaneous imaging of the activity dynamics of three key nodes in the caspase network.…”

Section: New Approaches For Multiplexed Imaging With Fp-based Biosensorsmentioning

confidence: 99%

Maximizing the performance of protein-based fluorescent biosensors

Chai

Cheng

Nasu

et al. 2023

Biochemical Society Transactions

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Fluorescent protein (FP)-based biosensors are genetically encoded tools that enable the imaging of biological processes in the context of cells, tissues, or live animals. Though widely used in biological research, practically all existing biosensors are far from ideal in terms of their performance, properties, and applicability for multiplexed imaging. These limitations have inspired researchers to explore an increasing number of innovative and creative ways to improve and maximize biosensor performance. Such strategies include new molecular biology methods to develop promising biosensor prototypes, high throughput microfluidics-based directed evolution screening strategies, and improved ways to perform multiplexed imaging. Yet another approach is to effectively replace components of biosensors with self-labeling proteins, such as HaloTag, that enable the biocompatible incorporation of synthetic fluorophores or other ligands in cells or tissues. This mini-review will summarize and highlight recent innovations and strategies for enhancing the performance of FP-based biosensors for multiplexed imaging to advance the frontiers of research.

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CASPAM: A Triple-Modality Biosensor for Multiplexed Imaging of Caspase Network Activity

Cited by 6 publications

References 41 publications

Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies

Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies

Robust and unbiased estimation of the background distribution for automated quantitative imaging

Maximizing the performance of protein-based fluorescent biosensors

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