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We provide evidence of a local synaptic nanoenvironment in the brain extracellular space (ECS) lying within 500 nm of postsynaptic densities. To reveal this brain compartment, we developed a correlative imaging approach dedicated to thick brain tissue based on single-particle tracking of individual fluorescent single wall carbon nanotubes (SWCNTs) in living samples and on speckle-based HiLo microscopy of synaptic labels. We show that the extracellular space around synapses bears specific properties in terms of morphology at the nanoscale and inner diffusivity. We finally show that the ECS juxta-synaptic region changes its diffusion parameters in response to neuronal activity, indicating that this nanoenvironment might play a role in the regulation of brain activity.

The Roman period is ubiquitous in popular media and the public consciousness, featuring regularly in formal education programmes, on film and television, and in museum displays. Scholarship into th...

The movement of ribosomes on mRNA is often interrupted by secondary structures that present mechanical barriers and play a central role in translation regulation. We investigate how ribosomes couple their internal conformational changes with the activity of translocation factor EF-G to unwind mRNA secondary structures using high-resolution optical tweezers with single-molecule fluorescence capability. We find that hairpin opening occurs during EF-G-catalyzed translocation and is driven by the forward rotation of the small subunit head. Modulating the magnitude of the hairpin barrier by force shows that ribosomes respond to strong barriers by shifting their operation to an alternative 7-fold-slower kinetic pathway prior to translocation. Shifting into a slow gear results from an allosteric switch in the ribosome that may allow it to exploit thermal fluctuations to overcome mechanical barriers. Finally, we observe that ribosomes occasionally open the hairpin in two successive sub-codon steps, revealing a previously unobserved translocation intermediate.

The encapsulation of enzymes and other proteins within a proteinaceous shell has been observed in many bacteria and archaea, but the function and utility of many such compartments are enigmatic. Efforts to study these functions have been complicated by the size and complexity of traditional protein compartments. One potential system for investigating the effect of compartmentalization is encapsulin, a large and newly discovered class of protein shells that are typically composed of two proteins: a protomer that assembles into the icosahedral shell and a cargo protein packaged inside. Encapsulins are some of the simplest known protein shell systems and readily self-assemble in vivo. Systematic characterization of the effects of compartmentalization requires the ability to load a wide range of cargo proteins. Here, we demonstrate that foreign cargo can be loaded into the encapsulin from Thermotoga maritima both in vivo and in vitro by fusion of the cargo protein with a short C-terminal peptide present in the native cargo. To facilitate biochemical characterization, we also develop a simple and rapid purification protocol and demonstrate the thermal and pH stability of the shell. Efforts to study the biophysical effects of protein encapsulation have been problematic in complex compartments, but the simplicity of assembling and loading encapsulin makes it an ideal system for future experiments exploring the effects of encapsulation on proteins.

We present a single molecule method for counting proteins within a diffraction-limited area when using photoactivated localization microscopy. The intrinsic blinking of photoactivatable fluorescent proteins mEos2 and Dendra2 leads to an overcounting error, which constitutes a major obstacle for their use as molecular counting tags. Here, we introduce a kinetic model to describe blinking and show that Dendra2 photobleaches three times faster and blinks seven times less than mEos2, making Dendra2 a better photoactivated localization microscopy tag than mEos2 for molecular counting. The simultaneous activation of multiple molecules is another source of error, but it leads to molecular undercounting instead. We propose a photoactivation scheme that maximally separates the activation of different molecules, thus helping to overcome undercounting. We also present a method that quantifies the total counting error and minimizes it by balancing over- and undercounting. This unique method establishes that Dendra2 is better for counting purposes than mEos2, allowing us to count in vitro up to 200 molecules in a diffraction-limited spot with a bias smaller than 2% and an uncertainty less than 6% within 10 min. Finally, we demonstrate that this counting method can be applied to protein quantification in vivo by counting the bacterial flagellar motor protein FliM fused to Dendra2.

International audience; The brain extracellular space (ECS) is a system of narrow compartments whose intricate nanometric structure has remained elusive until very recently. Understanding such a complex organisation represents a technological challenge that requires a technique able to resolve these nanoscopic spaces and simultaneously characterize their rheological properties. We recently used single-walled carbon nanotubes (SWCNTs) as near-infrared fluorescent probes to map with nanoscale precision the local organization and rheology of the ECS. Here we expand our method by tracking single nanotubes through super-resolution imaging in rat organotypic hippocampal slices and acute brain slices from adult mice, pioneering the exploration of the adult brain ECS at the nanoscale. We found a highly heterogeneous ECS, where local rheological properties can change drastically within few nanometres. Our results suggest differences in local ECS diffusion environments in organotypic slices when compared to adult mouse slices. Data obtained from super-resolved maps of the SWCNT trajectories indicate that ECS widths may vary between brain tissue models, with a looser, less crowded nano-environment in organotypic cultured slices.