Cancer cells express tumour-specific antigens derived via genetic and epigenetic alterations, which may be targeted by T-cell-mediated immune responses. However, cancer cells can avoid immune ...surveillance by suppressing immunity through activation of specific inhibitory signalling pathways, referred to as immune checkpoints. In recent years, the blockade of checkpoint molecules such as PD-1, PD-L1 and CTLA-4, with monoclonal antibodies has enabled the development of breakthrough therapies in oncology, and four therapeutic antibodies targeting these checkpoint molecules have been approved by the FDA for the treatment of several types of cancer. Here, we report the crystal structures of checkpoint molecules in complex with the Fab fragments of therapeutic antibodies, including PD-1/pembrolizumab, PD-1/nivolumab, PD-L1/BMS-936559 and CTLA-4/tremelimumab. These complex structures elucidate the precise epitopes of the antibodies and the molecular mechanisms underlying checkpoint blockade, providing useful information for the improvement of monoclonal antibodies capable of attenuating checkpoint signalling for the treatment of cancer.
Optogenetic approaches for controlling Ca2+ channels provide powerful means for modulating diverse Ca2+-specific biological events in space and time. However, blue light-responsive photoreceptors ...are, in principle, considered inadequate for deep tissue stimulation unless accompanied by optic fiber insertion. Here, we present an ultra-light-sensitive optogenetic Ca2+ modulator, named monSTIM1 encompassing engineered cryptochrome2 for manipulating Ca2+ signaling in the brain of awake mice through non-invasive light delivery. Activation of monSTIM1 in either excitatory neurons or astrocytes of mice brain is able to induce Ca2+-dependent gene expression without any mechanical damage in the brain. Furthermore, we demonstrate that non-invasive Ca2+ modulation in neurons can be sufficiently and effectively translated into changes in behavioral phenotypes of awake mice.Optogenetic applications in the brain of live animals often require the use of optic fibers due to poor tissue-penetration of blue light. Here the authors present monSTIM1, an improved high sensitivity optogenetic tool able to modulate Ca2+ signaling in the brain of awake mice using non-invasive light stimulation.
Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted ...as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.
The PFC is thought to be the region where remote memory is recalled. However, the neurotrophic receptors that underlie the remote memory remain largely unknown. Here, we benefited from auto-assembly ...split Cre to accomplish the neural projection-specific recombinase activity without spontaneous leakage. Deletion of tropomyosin receptor kinase B (TrkB) in neurons projecting from the medial entorhinal cortex to the mPFC displayed reduced remote memory recall from the male mice, but the recent recall was intact. We found that the TrkB deletion attenuates the participation of mPFC cells in the remote fear memory recall. The disruption of remote recall was attributed to reduced reactivation of cells in the mPFC. Notably, TrkB deletion seriously inhibited experience-dependent maturation of oligodendroglia in the PFC, resulting in defects in remote recall that were rescued by clemastine administration. Together, our data suggest that TrkB in intercortical circuits functions in remote memory consolidation.
Retrieving the past experiences or events is essential for the ones to lead life. The investigations performed in the rodent model have disclosed that the systems consolidation of memory accompanying changes of cortical circuits and transcriptome is required for maintaining the memory for a long time. In this study, the split Cre with TrkB
mice were subjected to discover that TrkB in the neurons plays a role in remote memory consolidation. We evaluated the contextual fear memory and labeled cells, which revealed deletion of TrkB interrupts newborn oligodendrocyte and reactivation of cells in mPFC at remote recall. Our data provide the implication that remote memory is relevant to neurotrophic receptor signaling as well as its influence on non-neuronal cells.
Phosphoinositide 3-kinases (PI3Ks) and Ras and Rho family small GTPases are key regulators of cell polarization, motility, and chemotaxis. They influence each other's activities by direct and ...indirect feedback processes that are only partially understood. Here, we show that 21 small GTPase homologs activate PI3K. Using a microscopy-based binding assay, we show that K-Ras, H-Ras, and five homologous Ras family small GTPases function upstream of PI3K by directly binding the PI3K catalytic subunit, p110. In contrast, several Rho family small GTPases activated PI3K by an indirect cooperative positive feedback that required a combination of Rac, CDC42, and RhoG small GTPase activities. Thus, a distributed network of Ras and Rho family small GTPases induces and reinforces PI3K activity, explaining past challenges to elucidate the specific relevance of different small GTPases in regulating PI3K and controlling cell polarization and chemotaxis.
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► Twenty-one small GTPases activate the PI3K pathway ► Upstream small GTPases activate PI3K via direct interaction ► Cooperation of downstream small GTPases is required for effective activation of PI3K
Intracellular antibodies have become powerful tools for imaging, modulating and neutralizing endogenous target proteins. Here, we describe an optogenetically activated intracellular antibody ...(optobody) consisting of split antibody fragments and blue-light inducible heterodimerization domains. We expanded this optobody platform by generating several optobodies from previously developed intracellular antibodies, and demonstrated that photoactivation of gelsolin and β2-adrenergic receptor (β2AR) optobodies suppressed endogenous gelsolin activity and β2AR signaling, respectively.
Ras and Rho small GTPases are critical for numerous cellular processes including cell division, migration, and intercellular communication. Despite extensive efforts to visualize the spatiotemporal ...activity of these proteins, achieving the sensitivity and dynamic range necessary for in vivo application has been challenging. Here, we present highly sensitive intensiometric small GTPase biosensors visualizing the activity of multiple small GTPases in single cells in vivo. Red-shifted sensors combined with blue light-controllable optogenetic modules achieved simultaneous monitoring and manipulation of protein activities in a highly spatiotemporal manner. Our biosensors revealed spatial dynamics of Cdc42 and Ras activities upon structural plasticity of single dendritic spines, as well as a broad range of subcellular Ras activities in the brains of freely behaving mice. Thus, these intensiometric small GTPase sensors enable the spatiotemporal dissection of complex protein signaling networks in live animals.
Despite efforts to visualize the spatio-temporal dynamics of single messenger RNAs, the ability to precisely control their function has lagged. This study presents an optogenetic approach for ...manipulating the localization and translation of specific mRNAs by trapping them in clusters. This clustering greatly amplified reporter signals, enabling endogenous RNA-protein interactions to be clearly visualized in single cells. Functionally, this sequestration reduced the ability of mRNAs to access ribosomes, markedly attenuating protein synthesis. A spatio-temporally resolved analysis indicated that sequestration of endogenous β-actin mRNA attenuated cell motility through the regulation of focal-adhesion dynamics. These results suggest a mechanism highlighting the indispensable role of newly synthesized β-actin protein for efficient cell migration. This platform may be broadly applicable for use in investigating the spatio-temporal activities of specific mRNAs in various biological processes.
Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient ...photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode. In addition, PA-Flp can be extended to the Cre-lox system through a viral vector as Flp-dependent Cre expression platform, thereby activating both Flp and Cre. Finally, we demonstrate that PA-Flp-dependent, Cre-mediated Ca
3.1 silencing in the medial septum increases object-exploration behavior in mice. Thus, PA-Flp is a noninvasive, highly efficient, and easy-to-use optogenetic module that offers a side-effect-free and expandable genetic manipulation tool for neuroscience research.
Secreted Wnts play crucial roles in synaptogenesis and synapse maintenance, but endogenous factors promoting synapse elimination in central neurons remain unknown. Here we show that proline-rich 7 ...(PRR7) induces specific removal of excitatory synapses and acts as a Wnt inhibitor. Remarkably, transmembrane protein PRR7 is activity-dependently released by neurons via exosomes. Exosomal PRR7 is uptaken by neurons through membrane fusion and eliminates excitatory synapses in neighboring neurons. Conversely, PRR7 knockdown in sparse neurons greatly increases excitatory synapse numbers in all surrounding neurons. These non-cell autonomous effects of PRR7 are effectively negated by augmentation or blockade of Wnt signaling. PRR7 exerts its effect by blocking the exosomal secretion of Wnts, activation of GSK3β, and promoting proteasomal degradation of PSD proteins. These data uncover a proximity-dependent, reciprocal mechanism for the regulation of excitatory synapse numbers in local neurons and demonstrate the significance of exosomes in inter-neuronal signaling in the vertebrate brain.
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