Imaging burst kinetics and spatial coordination during serial killing by single natural killer cells

Choi, Paul J.; Mitchison, Timothy J.

doi:10.1073/pnas.1221312110

Cited by 107 publications

(124 citation statements)

References 38 publications

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“…However, recent studies proposed that lytic granule secretion may not even require prior polarization of the centrosome (24). Our data describing the sequential delivery of lethal hits with adjacent targets are consistent with a recently published study that investigated the kinetics of serial killing using the NK cell line, NK-92-MI (25). The authors reported that most serial killing events involved killing of adjacent targets, and they proposed that detachment from one target cell is directly regulated by subsequent attachment to the following target cell.…”

Section: Discussionsupporting

confidence: 91%

Rapid and Unidirectional Perforin Pore Delivery at the Cytotoxic Immune Synapse

Lopez

Jenkins

Rudd-Schmidt

et al. 2013

The Journal of Immunology

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The effective engagement of cytotoxic lymphocytes (CLs) with their target cells is essential for the removal of virus-infected and malignant cells from the body. The spatiotemporal properties that define CL engagement and killing of target cells remain largely uncharacterized due to a lack of biological reporters. We have used a novel live cell microscopy technique to visualize the engagement of primary human and mouse CL with their targets and the subsequent delivery of the lethal hit. Extensive quantitative real-time analysis of individual effector–target cell conjugates demonstrated that a single effector calcium flux event was sufficient for the degranulation of human CLs, resulting in the breach of the target cell membrane by perforin within 65–100 s. In contrast, mouse CLs demonstrated distinct calcium signaling profiles leading to degranulation: whereas mouse NKs required a single calcium flux event, CD8+ T cells typically required several calcium flux events before perforin delivery. Irrespective of their signaling profile, every target cell that was damaged by perforin died by apoptosis. To our knowledge, we demonstrate for the first time that perforin pore delivery is unidirectional, occurring exclusively on the target cell membrane, but sparing the killer cell. Despite this, the CTL membrane was not intrinsically perforin resistant, as intact CTLs presented as targets to effector CTLs were capable of being killed by perforin-dependent mechanisms. Our results highlight the remarkable efficiency and specificity of perforin pore delivery by CLs.

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Section: Discussionsupporting

confidence: 91%

Rapid and Unidirectional Perforin Pore Delivery at the Cytotoxic Immune Synapse

Lopez

Jenkins

Rudd-Schmidt

et al. 2013

The Journal of Immunology

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“…To confirm that GB enters the mitochondria, a fluorescence resonance energy transfer (FRET)-based approach was used. HeLa cells were transfected with a GB-FRET reporter consisting of ECFP connected to EYFP with a linker containing a GB cleavage site VGPDFGR 29 that was targeted either to the cytosol or to the mitochondrial matrix (GB-cyto-FRET and GB-mito-FRET) (Supplementary Figures 1A and B). GB and perforin (P) treatment triggered a loss of FRET signal from the mito-FRET reporter with a similar kinetic to that of the cytosolic reporter, indicating that GB entered the mitochondrial matrix (Figures 1a-c and Supplementary Figures 1A and B).…”

Section: Resultsmentioning

confidence: 99%

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis

et al. 2017

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We have found that granzyme B (GB)-induced apoptosis also requires reactive oxygen species resulting from the alteration of mitochondrial complex I. How GB, which does not possess a mitochondrial targeting sequence, enter this organelle is unknown. We show that GB enters the mitochondria independently of the translocase of the outer mitochondrial membrane complex, but requires instead Sam50, the central subunit of the sorting and assembly machinery that integrates outer membrane β-barrel proteins. Moreover, GB breaches the inner membrane through Tim22, the metabolite carrier translocase pore, in a mitochondrial heat-shock protein 70 (mtHsp70)-dependent manner. Granzyme A (GA) and caspase-3 use a similar route to the mitochondria. Finally, preventing GB from entering the mitochondria either by mutating lysine 243 and arginine 244 or depleting Sam50 renders cells more resistant to GB-mediated reactive oxygen species and cell death. Similarly, Sam50 depletion protects cells from GA-, GM-and caspase-3-mediated cell death. Therefore, cytotoxic molecules enter the mitochondria to induce efficiently cell death through a noncanonical Sam50-, Tim22-and mtHsp70-dependent import pathway.Cytotoxic lymphocytes eradicate pathogen-infected or transformed cells mainly through the cytotoxic granule pathway. [1][2][3][4] Among the five human granzymes (A, B, H, K and M), granzyme B (GB) triggers cell death in both a caspasedependent and caspase-independent manner, although human and mouse GB differ in their requirement for caspase activation. 5-10 GB-induced cell death also involves Bid/Bax/ Bak-mediated mitochondrial outer membrane permeabilization for the release of the apoptogenic factors cytochrome c, Smac/Diablo, Endo G, HtrA2 and AIF. 1,9-17 We have recently reported that mitochondrial reactive oxygen species (ROS) have a critical role in GB pathway by amplifying apoptogenic factor release, oligonucleosomal DNA fragmentation and lysosomal membrane rupture. 18 Mitochondrial ROS resulted from GB-mediated cleavage of NDUFV1, NDUFS1 and NDUFS2, three subunits of mitochondrial complex I. 18 How GB that does not possess a mitochondrial targeting sequence enters the mitochondria is not known. The mitochondrial nucleoid encodes only for 13 structural proteins, while the rest of the mitochondrial proteome is nuclear-encoded and transported to the mitochondria through a sophisticated protein import machinery. [19][20][21][22][23][24][25][26][27][28] The translocase of the outer mitochondrial membrane (TOM), the entry gate to the mitochondria, 20 passes on the cargos to the sorting and assembly machinery (SAM) complex, to the mitochondrial intermembrane space assembly (MIA) complex and to the translocases of the inner mitochondrial membrane TIM22 or TIM23 complexes for delivery to the outer membrane, the intermembrane space, the inner membrane or the matrix, respectively. [19][20][21][22]25,26,28 Unexpectedly, we found that GB mitochondrial entry is independent of the TOM-TIM23 complexes, but requires instead the Sam50 and Tim22 chann...

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“…4B; Conway et al, 2014Conway et al, , 2017Newman et al, 2011). Applications in cell proliferation (Harvey et al, 2008;Mochizuki et al, 2001), survival (Onuki et al, 2002;Tyas et al, 2000), signalling (Shih and Qi, 2017;Weitsman et al, 2016), immune cell activity (Choi and Mitchison, 2013;Li et al, 2016b), metabolism (Fehr et al, 2003;Imamura et al, 2009;Mächler et al, 2016;Okumoto et al, 2005) or migration (Seong et al, 2011;Wang et al, 2005) with a recent move to generate GEMMs of FRET biosensors for high-fidelity intravital imaging (Johnsson et al, 2014;Kamioka et al, 2012;Mukherjee et al, 2016;Nobis et al, 2017).…”

Section: Box 1 Subcellular Imaging Techniquesmentioning

confidence: 99%

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

Nobis

Warren

Lucas

et al. 2018

Journal of Cell Science

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Molecular mobility, localisation and spatiotemporal activity are at the core of cell biological processes and deregulation of these dynamic events can underpin disease development and progression. Recent advances in intravital imaging techniques in mice are providing new avenues to study real-time molecular behaviour in intact tissues within a live organism and to gain exciting insights into the intricate regulation of live cell biology at the microscale level. The monitoring of fluorescently labelled proteins and agents can be combined with autofluorescent properties of the microenvironment to provide a comprehensive snapshot of in vivo cell biology. In this Review, we summarise recent intravital microscopy approaches in mice, in processes ranging from normal development and homeostasis to disease progression and treatment in cancer, where we emphasise the utility of intravital imaging to observe dynamic and transient events in vivo. We also highlight the recent integration of advanced subcellular imaging techniques into the intravital imaging pipeline, which can provide in-depth biological information beyond the singlecell level. We conclude with an outlook of ongoing developments in intravital microscopy towards imaging in humans, as well as provide an overview of the challenges the intravital imaging community currently faces and outline potential ways for overcoming these hurdles.

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Imaging burst kinetics and spatial coordination during serial killing by single natural killer cells

Cited by 107 publications

References 38 publications

Rapid and Unidirectional Perforin Pore Delivery at the Cytotoxic Immune Synapse

Rapid and Unidirectional Perforin Pore Delivery at the Cytotoxic Immune Synapse

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

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