Remodelling of Cortical Actin Where Lytic Granules Dock at Natural Killer Cell Immune Synapses Revealed by Super-Resolution Microscopy

Brown, Alice C. N.; Oddos, Stéphane; Dobbie, Ian M.; Alakoskela, Juha-Matti; Parton, Richard M.; Eissmann, Philipp; Neil, Mark A. A.; Dunsby, Christopher; French, Paul M. W.; Davis, Ilan; Davis, Daniel M.

doi:10.1371/journal.pbio.1001152

Cited by 212 publications

(293 citation statements)

References 75 publications

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“…In human CTLs, it was found that the MTOC did not enter in the TIRF plane (18); conversely, in mouse NK cells, it was found that MTOC appeared in the TIRF plane and lytic granules mostly associated with MTOC (19). In line with the latter observation, superresolution microscopy analysis of the NK secretory domain showed that MTOC polarizes toward the granule penetrable synaptic areas of the actin meshwork (20).…”

Section: Discussionmentioning

confidence: 74%

An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse

Bertrand

Müller

Roh

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

105

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It is presently assumed that lethal hit delivery by cytotoxic T lymphocytes (CTLs) is mechanistically linked to centrosome polarization toward target cells, leading to dedicated release of lytic granules within a confined secretory domain. Here we provide three lines of evidence showing that this mechanism might not apply as a general paradigm for lethal hit delivery. First, in CTLs stimulated with immobilized peptide-MHC complexes, lytic granules and microtubule organizing center localization into synaptic areas are spatio-temporally dissociated, as detected by total internal reflection fluorescence microscopy. Second, in many CTL/target cell conjugates, lytic granule secretion precedes microtubule polarization and can be detected during the first minute after cell-cell contact. Third, inhibition of microtubule organizing center and centrosome polarization impairs neither lytic granule release at the CTL synapse nor killing efficiency. Our results broaden current views of CTL biology by revealing an extremely rapid step of lytic granule secretion and by showing that microtubule organizing center polarization is dispensable for efficient lethal hit delivery.immunological synapse | T-cell antigen receptor | protein kinase Cζ | signal transduction

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Section: Discussionmentioning

confidence: 74%

An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse

Bertrand

Müller

Roh

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

105

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“…Our results suggest that recovered cortical actin may act as a barrier to limit granule secretion during a discrete CTL-target encounter and, furthermore, that recovery itself is triggered upon granule fusion, providing a mechanism that facilitates serial killing during an immune response. We also show that both clearance and recovery of cortical actin occurs concomitantly with complementary changes in PIP 2 , suggesting that The role of actin in secretion has been studied in many cell types specializing in exocytosis, including natural killer (NK) cells (35)(36)(37), mast cells (7), pancreatic β cells (38), and adrenal chromaffin cells (39,40). One model, known as the "barrier model," suggests that the actin cortex acts physically to prevent vesicles from coming in close enough proximity to the plasma membrane for fusion to occur (38).…”

Section: Discussionmentioning

confidence: 75%

Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes

Ritter

Kapnick

Murugesan

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

102

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CD8 + cytotoxic T lymphocytes (CTLs) eliminate virally infected cells through directed secretion of specialized lytic granules. Because a single CTL can kill multiple targets, degranulation must be tightly regulated. However, how CTLs regulate the termination of granule secretion remains unclear. Previous work demonstrated that centralized actin reduction at the immune synapse precedes degranulation. Using a combination of live confocal, total internal reflection fluorescence, and superresolution microscopy, we now show that, after granule fusion, actin recovers at the synapse and no further secretion is observed. Depolymerization of actin led to resumed granule secretion, suggesting that recovered actin acts as a barrier preventing sustained degranulation. Furthermore, RAB27a-deficient CTLs, which do not secrete cytotoxic granules, failed to recover actin at the synapse, suggesting that RAB27a-mediated granule secretion is required for actin recovery. Finally, we show that both actin clearance and recovery correlated with synaptic phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and that alterations in PIP 2 at the immunological synapse regulate cortical actin in CTLs, providing a potential mechanism through which CTLs control cortical actin density. Our work provides insight into actin-related mechanisms regulating CTL secretion that may facilitate serial killing during immune responses.cytotoxic T lymphocytes | actin | lytic granule secretion | PIP 2 | RAB27a

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“…A number of distinct lymphocyte activating receptors are known to form microclusters in response to ligand binding (16)(17)(18)(19). It is generally thought that this clustering facilitates signal transduction by providing activating receptors and receptor proximal enzymes privileged access to their substrates and binding partners while at the same time excluding negative regulators.…”

mentioning

confidence: 99%

Protein Kinase C-θ Clustering at Immunological Synapses Amplifies Effector Responses in NK Cells

Merino

Abeyweera

Firth

et al. 2012

The Journal of Immunology

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In lymphocytes, stimulation of cell surface activating receptors induces the formation of protein microclusters at the plasma membrane that contain the receptor itself, along with other signaling molecules. Although these microclusters are generally thought to be crucial for promoting downstream cellular responses, evidence that specifically links clustering potential to signaling output is lacking. We found that protein kinase C-θ (PKCθ), a key signaling molecule in multiple lymphocyte subsets, formed microclusters in activated NK cells. These microclusters coalesced within the immunological synapse between the NK cell and its target cell. Clustering was mediated by the regulatory region of PKCθ and specifically required a putative phosphotyrosine-binding site within its N-terminal C2 domain. Whereas expression of wild-type PKCθ rescued the cytokine production defect displayed by PKCθ-deficient NK cells, expression of a PKCθ point-mutant incapable of forming microclusters had little to no effect. Hence, PKCθ clustering was necessary for optimal effector function. Notably, only receptors containing ITAMs induced PKCθ microclusters on their own, explaining previous observations that ITAM-coupled receptors promote stronger activating signals and effector responses than do receptors lacking these motifs. Taken together, our results provide a cell biological basis for the role of PKCθ clustering during NK cell activation, and highlight the importance of subcellular compartmentalization for lymphocyte signal transduction.

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Remodelling of Cortical Actin Where Lytic Granules Dock at Natural Killer Cell Immune Synapses Revealed by Super-Resolution Microscopy

Abstract: Super-resolution 3D imaging reveals remodeling of the cortical actin meshwork at the natural killer cell immune synapse, which is likely to be important for secretion of lytic granules.

Cited by 212 publications

References 75 publications

An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse

An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse

Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes

Protein Kinase C-θ Clustering at Immunological Synapses Amplifies Effector Responses in NK Cells

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