Selective induction of targeted cell death and elimination by near-infrared femtosecond laser ablation

Abstract

The techniques for inducing the death of specific cells in tissue has attracted attention as new methodologies for studying cell function and tissue regeneration. In this study, we show that a sequential process of targeted cell death and removal can be triggered by short-term exposure of near-infrared femtosecond laser pulses. Kinetic analysis of the intracellular accumulation of trypan blue and the assay of caspase activity revealed that femtosecond laser pulses induced immediate disturbance of plasma membrane integrity followed by apoptosis-like cell death. Yet, adjacent cells showed no sign of membrane damage and no increased caspase activity. The laser-exposed cells eventually detached from the substrate after a delay of >54 min while adjacent cells remained intact. On the base of in vitro experiments, we applied the same approach to ablate targeted single cardiac cells of a live zebrafish heart. The ability of inducing targeted cell death with femtosecond laser pulses should find broad applications that benefit from precise cellular manipulation at the level of single cells in vivo and in vitro .

Optogenetic activators of apoptosis, necroptosis and pyroptosis for probing cell death dynamics and bystander cell responses

“…This is in line with reports showing that caspase-1 activation in Gsdmd -deficient mouse macrophages engages rapid apoptosis via Bid cleavage 14–16 . Of note, while mouse caspase-11 is apparently not able to induce this type of apoptosis 7–9 , we found that optoCaspase-4 and −5 caused GSDMD-independent apoptosis, which could possibly be due to their wider substrate profile 17 . OptoCaspase activation efficiently induced pyroptosis in commonly used laboratory cell lines, such as MCF7 cells, HeLa, Caco-2, HT-29 and HaCaT, ( Fig.…”

“…Whereas other laser-based methods (such as laser ablation, laser-induced DNA-damage or photosensitizers, such as KillerRed) can also be used to induce cell death rapidly, in selected cells and in vivo [41][42][43][44][45] , they often fail to elicit specific cell death modes, rather relying on less specific types of cellular damage 46 . Laser ablation for example induces mixed apoptotic and necrotic phenotypes 7,8 , which limits its application to study the response of cells or tissue to specific types of PCD. By contrast, the optoCDE approach enables highly specific activation of selected cell death programs directly at effector protein level, increasing the specificity of cell death program and reducing the impact of endogenous regulation and inter-pathway crosstalk.…”

“…To highlight the usefulness and versatility of the novel tools for the study of complex biological questions, we compared the fates of cells dying by lytic (pyroptosis or necroptosis) versus non-lytic (apoptotic) cell death in the context of confluent epithelial monolayers. Previous studies reported that apoptosis is followed by extrusion of dead cells from monolayers, but death was usually induced non-specifically by laser ablation that causes a mixed apoptotic/necrotic phenotype [6][7][8] and fates of necroptotic/pyroptotic cells have not been studied on a single cell level before.…”

“…The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101/2021.08.31.458313 doi: bioRxiv preprint 3 analyses, such as the low specificity of cell death-inducing stimuli, which affect both the targeted cell as well as its neighbors, the crosstalk between different PCD pathways, and the difficulty to induce death in a spatially and temporally controlled manner. One strategy to overcome these obstacles is the use of laser-induced ablation of single or multiple cells [6][7][8] , as it allows to study the signaling response of direct neighbors and the extrusion of dead cells. However, it remains unclear what type of death is caused by laser ablation, since dying cells often display the features of both apoptosis (caspase activation) and necrosis (membrane permeabilization) 6 .…”

Optogenetic activators of apoptosis, necroptosis, and pyroptosis

“…Experimentally, such analyses are hindered by the low specificity of cell death-inducing stimuli, which affect both the targeted cell and its neighbors, the crosstalk between different PCD pathways, and the difficulty to induce death in a spatially and temporally controlled manner. Laser-induced ablation of single or multiple cells ( Okano et al, 2020 ; Tirlapur et al, 2001 ; Uchugonova et al, 2008 ) has been used to overcome these obstacles, as it allows to study the signaling response of direct neighbors and dead cell extrusion. However, it is unclear what type of death is caused by laser ablation since dying cells often display features of both apoptosis (caspase activation) and necrosis (membrane permeabilization; Tirlapur et al, 2001 ).…”