Live Cell Linear Dichroism Imaging Reveals Extensive Membrane Ruffling within the Docking Structure of Natural Killer Cell Immune Synapses

Benninger, Richard K.P.; Vanherberghen, Bruno; Young, Stephen; Taner, Sabrina B.; Culley, Fiona J.; Schnyder, Tim; Neil, Mark A. A.; Wüstner, Daniel; French, Paul M. W.; Davis, Daniel M.; Önfelt, Björn

doi:10.1016/j.bpj.2008.10.005

Cited by 30 publications

(35 citation statements)

References 11 publications

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“…Microscopic examination of monocytes revealed dramatic ruffling of the plasma membrane upon addition of GPN (Fig. 2D), characteristic of exocytosis described in other cell types (Benninger et al, 2009). We next tested the dependency of GPN-evoked calcium response on NEM.…”

Section: Gpn and Thapsigargin Triggers Lysosomes Exocytosis In Monocytesmentioning

confidence: 77%

Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes

Sivaramakrishnan

Bidula

Campwala

et al. 2012

Journal of Cell Science

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Section: Gpn and Thapsigargin Triggers Lysosomes Exocytosis In Monocytesmentioning

confidence: 77%

Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes

Sivaramakrishnan

Bidula

Campwala

et al. 2012

Journal of Cell Science

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“…24,26,27 Membrane intercalation in cell conjugates has been described to happen in cytotoxic NK-cell synapses. 28 Indeed, this event could lead to a misevaluation of the cellular contribution for f-actin enrichment at the synapse, considering the resolution of 60 nm in light microscopy used in our study. Therefore, we studied the morphology of DC/NK-cell conjugates by transmission electron microscopy (TEM).…”

Section: Resultsmentioning

confidence: 99%

Cytoskeletal stabilization of inhibitory interactions in immunologic synapses of mature human dendritic cells with natural killer cells

Silva

Graf

Münz

2011

Blood

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IntroductionDendritic cells (DCs) are key players in the immune system as they bridge innate and adaptive immunity. 1 In their immature form, DCs reside as sentinels for pathogens and stress signals in almost all organs of the human body. On interaction with so-called maturation stimuli, DCs migrate at increased frequency to secondary lymphoid organs for the initiation of immune responses. 2 DC maturation leads also to the up-regulation of antigen presentation on MHC molecules, 3 of chemokine receptors and of costimulatory molecules, 4 as well as to the release of cytokines. 5,6 In secondary lymphoid tissues, mature DCs are able to alert and activate cells of the adaptive immune system, like T cells, and also of the innate immune system, like natural killer (NK) cells. [7][8][9] Indeed, NK-cell activation by DCs is required for many immune responses. [10][11][12][13][14][15] On interaction with mature DCs and the cytokines that they produce, resting NK cells, preferentially those resident in secondary lymphoid organs, secrete IFN-␥, TNF, and GM-CSF. This effect is mostly mediated via IL-12 and IL-18 production by DCs. 7,[16][17][18] Moreover, cytokines secreted by DC-activated NK cells induce further maturation of DCs in secondary lymphoid tissues, prompting them to efficiently stimulate CTL responses. Furthermore, type I IFN of mature DCs up-regulates cytotoxicity of NK cells for powerful anti-tumor 19 and anti-viral immune responses. 17,20 Finally, the interaction with mature DCs also elicits resting NK-cell priming, 10 survival 21,22 and proliferation, 7 via DC produced IL-15. Thus, mature DCs secrete several cytokines that stimulate distinct NK-cell functions.Up-regulation of NK-cell cytotoxicity through this interaction could lead to the killing of DCs, thereby compromising the priming of efficient adaptive immune control by these antigen presenting cells. To minimize DC lysis, mature DCs have developed mechanisms that prevent the cytotoxic effect of activated lymphocytes, while immature DCs can be edited by activated NK cells via NKp30 mediated recognition. 3 Among these protective mechanisms against cell-mediated cytotoxicity, mature DCs express members of the serpin-family of serin protease-inhibitors that prevent apoptosis induction by granzyme B. 23 Moreover, maturation leads to the up-regulation of MHC class I molecules on the surface of DCs. MHC class I molecules interact with inhibitory receptors on the NK-cell surface, controlling the activation of these lymphocytes. 3 Thus, a balance between activating and inhibitory signals seems to exist at the immunologic synapse between mature DCs and resting NK cells. 21 In this way, resting NK cells can be efficiently activated by DCs and, at the same time, mature DCs are protected from being killed.To characterize the development of the regulatory synapse between mature DCs and resting NK cells, we describe here the kinetics of distribution of cytoskeletal elements, as well as activating and inhibitory molecules in conjugates of mature DCs with resting NK c...

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“…Molecules act as oriented absorption/emission dipoles, whose pointing direction can be monitored, exploiting the polarized nature of light. This property has been used to determine the organization of molecular assemblies whose orientation is constrained, in particular in the cell membrane (7)(8)(9)(10)(11). Recent works using polarized microscopy have also evidenced microscopic-scale organization of septin filaments in budding yeast (12,13) and actin filaments in Drosophila tissues (14), opening a path for in vivo structural imaging.…”

mentioning

confidence: 99%

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

Valades-Cruz

Shaban

Kress

et al. 2016

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

126

138

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Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.iological processes are inherently driven by the molecularscale organization of biomolecular assemblies, which arrange in precise structures that are essential for biological functions in cells and tissues. The extent to which the biological function depends on the underlying molecular-scale organization is particularly evident in filamentous assemblies, such as DNA filaments and cytoskeletal protein filaments. Changes in the local higher-order organization of DNA filaments is tightly linked to essential DNA-mediated processes including control of gene expression, DNA replication, and DNA repair. However, how specific DNA-binding proteins affect DNA filament architecture and thus DNA-mediated functions is poorly understood (1). Similarly, the spatial organization of cytoskeletal filaments in cells and tissues is also weakly explored, despite their central role in generating forces and driving cell motility, cell division, and tissue morphogenesis (2). Electron microscopy has been widely used to provide molecular-scale images of the structure of such filament assemblies; however, it typically involves several daylong sample preparation and ultrathin sectioning of the biological material, thus limiting investigations in whole cells and tissues.Polarized fluorescence imaging is a powerful approach for elucidating the structural organization of filament assemblies because it is compatible with a wide variety of microscopy techniques, thus enabling studies across multiple spatial and temporal scales. Polarized fluorescenc...

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Live Cell Linear Dichroism Imaging Reveals Extensive Membrane Ruffling within the Docking Structure of Natural Killer Cell Immune Synapses

Cited by 30 publications

References 11 publications

Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes

Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes

Cytoskeletal stabilization of inhibitory interactions in immunologic synapses of mature human dendritic cells with natural killer cells

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

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