Inflammation is fundamental for protecting the organism against infection and injury. However, a failure to control immune response results in chronic inflammation and several associated disorders such as pain and loss of function. Initiation of inflammation is orchestrated by cytokines, among which interleukin (IL)-1β is particularly important. IL-1β is synthesized as an inactive protein that has to be processed by the inflammasome to generate the mature bioactive form. Conventional techniques cannot monitor IL-1β activation with high spatial and temporal resolution. Here, we present a ratiometric biosensor that allows monitoring IL-1β processing in real-time, with a temporal resolution of seconds and with a single cell spatial resolution. Using this sensor, we describe for the first time the kinetic of the inflammasome activity in living macrophages. With this new probe we also demonstrated that the pro-IL-1β processing occurs all over the cytoplasm.

Synaptic activity–dependent remodeling of the glutamate receptor scaffold complex generates a negative feedback loop that limits further NMDA receptor activation.

Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor–mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.

Inflammation is fundamental for protecting the organism against infection and injury. However, a failure to control immune response results in chronic inflammation and several associated disorders such as pain and loss of function. Initiation of inflammation is orchestrated by cytokines, among which IL-1β is particularly important. IL-1β is synthesized as an inactive protein that has to be processed by the inflammasome to generate the mature bioactive form. Conventional techniques cannot monitor IL-1β activation with high spatial and temporal resolution. In this study, we present a ratiometric biosensor that allows monitoring IL-1β processing in real time, with a temporal resolution of seconds and with a single-cell spatial resolution. Using this sensor, to our knowledge, we describe for the first time the kinetic of the inflammasome activity in living macrophages. With this new probe, we also demonstrated that the pro–IL-1β processing occurs all over the cytoplasm.

Scaffolding proteins that are associated with glutamate receptors in dendritic spines govern the location and function of receptors to control synaptic transmission. Unraveling the spatio-temporal dynamics of protein-protein interactions within components of the scaffolding complex will bring to light the function of these interactions. Combining bioluminescence resonance energy transfer (BRET) imaging to electrophysiological recordings, we have recently shown that GKAP, a core protein of the scaffolding complex, interacts with DLC2, a protein associated with molecular motors. Synaptic activity-induced GKAP-DLC2 interaction in spines stabilizes the scaffolding complex and enhances the NMDA currents. Interestingly, this work placed emphasis on the bioarchitectural dependence of protein-protein interaction dynamics. Depending on physiological conditions, the modulation in space and time of protein-protein interaction is acutely regulated, engendering a subtle control of synaptic transmission in the state of the individual synapse.

Although G-protein coupled receptors (GPCRs) have been shown to assemble into functional homo or heteromers, the role of each protomer in G-protein activation is not known. Among the GPCRs, the γ-aminobutyric acid (GABA) type B receptor (GABABR) is the only one known so far that needs two subunits, GB1 and GB2, to function. The GB1 subunit contains the GABA binding site but is unable to activate G-proteins alone. In contrast the GB2 subunit which does not bind GABA, has an heptahelical domain able to activate G-proteins when assembled into dimers (Galvez et al., EMBO J. 20, 2001, 2152–2159). In the present study, we examined the role of each subunit within the GB1-GB2 heteromer, in G-protein coupling. To that aim, point mutations in the highly conserved third intracellular loop known to prevent G-protein activation of the related Ca-sensing or metabotropic glutamate receptors were introduced into GB1 and GB2. One mutation, L686P introduced in GB2 prevents the formation of a functional receptor, even though the heteromer reaches the cell surface, and even though the mutated subunit still associates with GB1 and increases GABA affinity on GB1. This was observed either in HEK293 cells where the activation of the G-protein was assessed by measurement of IP accumulation, or in cultured neurons where the inhibition of the Ca-channel current was measured. In contrast, the same mutation when introduced into GB1 does not modify the G-protein coupling properties of the heteromeric GABAB receptor either in HEK293 cells or in neurons. These data show that a single subunit in a dimeric GPCR is critical for coupling to G-proteins.