Dual Color Sensors for Simultaneous Analysis of Calcium Signal Dynamics in the Nuclear and Cytoplasmic Compartments of Plant Cells

Spatiotemporal changes in cellular calcium (Ca2+) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca2+ oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Forster resonance energy transfer (FRET) yellow-based Ca2+ cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca2+ signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca2+ changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca2+ sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca2+ signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenic Medicago truncatula roots and Arabidopsis thaliana were used to successfully monitor Ca2+ responses to microbial biotic and abiotic elicitors. In M. truncatula, we demonstrate that GECOs detect symbiosis-related Ca2+ spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in both M. truncatula and A. thaliana, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca2+ signaling in vivo. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca2+ signaling dynamics in vivo in response to different stimuli in multi-subcellular compartments of plant cells

Dual Color Sensors for Simultaneous Analysis of Calcium Signal Dynamics in the Nuclear and Cytoplasmic Compartments of Plant Cells

Spatiotemporal changes in cellular calcium (Ca2+) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca2+ oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca2+ cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca2+ signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca2+ changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca2+ sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca2+ signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenic Medicago truncatula roots and Arabidopsis thaliana were used to successfully monitor Ca2+ responses to microbial biotic and abiotic elicitors. In M. truncatula, we demonstrate that GECOs detect symbiosis-related Ca2+ spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in both M. truncatula and A. thaliana, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca2+ signaling in vivo. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca2+ signaling dynamics in vivo in response to different stimuli in multi-subcellular compartments of plant cells

Video1.AVI

<p>Spatiotemporal changes in cellular calcium (Ca²⁺) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca²⁺ oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca²⁺ cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca²⁺ signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca²⁺ changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca²⁺ sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca²⁺ signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenic Medicago truncatula roots and Arabidopsis thaliana were used to successfully monitor Ca²⁺ responses to microbial biotic and abiotic elicitors. In M. truncatula, we demonstrate that GECOs detect symbiosis-related Ca²⁺ spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in both M. truncatula and A. thaliana, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca²⁺ signaling in vivo. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca²⁺ signaling dynamics in vivo in response to different stimuli in multi-subcellular compartments of plant cells.</p

Video5.AVI

<p>Spatiotemporal changes in cellular calcium (Ca²⁺) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca²⁺ oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca²⁺ cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca²⁺ signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca²⁺ changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca²⁺ sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca²⁺ signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenic Medicago truncatula roots and Arabidopsis thaliana were used to successfully monitor Ca²⁺ responses to microbial biotic and abiotic elicitors. In M. truncatula, we demonstrate that GECOs detect symbiosis-related Ca²⁺ spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in both M. truncatula and A. thaliana, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca²⁺ signaling in vivo. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca²⁺ signaling dynamics in vivo in response to different stimuli in multi-subcellular compartments of plant cells.</p

DataSheet1.PDF

<p>Spatiotemporal changes in cellular calcium (Ca²⁺) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca²⁺ oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca²⁺ cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca²⁺ signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca²⁺ changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca²⁺ sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca²⁺ signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenic Medicago truncatula roots and Arabidopsis thaliana were used to successfully monitor Ca²⁺ responses to microbial biotic and abiotic elicitors. In M. truncatula, we demonstrate that GECOs detect symbiosis-related Ca²⁺ spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in both M. truncatula and A. thaliana, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca²⁺ signaling in vivo. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca²⁺ signaling dynamics in vivo in response to different stimuli in multi-subcellular compartments of plant cells.</p

Video4.AVI

<p>Spatiotemporal changes in cellular calcium (Ca²⁺) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca²⁺ oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca²⁺ cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca²⁺ signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca²⁺ changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca²⁺ sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca²⁺ signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenic Medicago truncatula roots and Arabidopsis thaliana were used to successfully monitor Ca²⁺ responses to microbial biotic and abiotic elicitors. In M. truncatula, we demonstrate that GECOs detect symbiosis-related Ca²⁺ spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in both M. truncatula and A. thaliana, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca²⁺ signaling in vivo. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca²⁺ signaling dynamics in vivo in response to different stimuli in multi-subcellular compartments of plant cells.</p

T cell migration, search strategies and mechanisms

3 figurasT cell migration is essential for T cell responses; it allows for the detection of cognate antigen at the surface of antigen-presenting cells and for interactions with other cells involved in the immune response. Although appearing random, growing evidence suggests that T cell motility patterns are strategic and governed by mechanisms that are optimized for both the activation stage of the cell and for environment-specific cues. In this Opinion article, we discuss how the combined effects of T cell-intrinsic and -extrinsic forces influence T cell motility patterns in the context of highly complex tissues that are filled with other cells involved in parallel motility. In particular, we examine how insights from 'search theory' can be used to describe T cell movement across an 'exploitation–exploration trade-off' in the context of activation versus effector function and lymph nodes versus peripheral tissuesThis work was supported by grants from US NIH (R01AI052116 and R01AI114787 to M.F.K.; R03AI119220 to A.G.) and the Human Frontier Science Program (RGY0084/2011 to F.B.).Peer reviewe