Enhanced incorporation of subnanometer tags into cellular proteins for fluorescence nanoscopy via optimized genetic code expansion.

With few-nanometer resolution recently achieved by a new generation of fluorescence nanoscopes (MINFLUX and MINSTED), the size of the tags used to label proteins will increasingly limit the ability to dissect nanoscopic biological structures. Bioorthogonal (click) chemical groups are powerful tools for the specific detection of biomolecules. Through the introduction of an engineered aminoacyl-tRNA synthetase/tRNA pair (tRNA: transfer ribonucleic acid), genetic code expansion allows for the site-specific introduction of amino acids with "clickable" side chains into proteins of interest. Well-defined label positions and the subnanometer scale of the protein modification provide unique advantages over other labeling approaches for imaging at molecular-scale resolution. We report that, by pairing a new N-terminally optimized pyrrolysyl-tRNA synthetase (chPylRS2020) with a previously engineered orthogonal tRNA, clickable amino acids are incorporated with improved efficiency into bacteria and into mammalian cells. The resulting enhanced genetic code expansion machinery was used to label ß-actin in U2OS cell filopodia for MINFLUX imaging with minimal separation of fluorophores from the protein backbone. Selected data were found to be consistent with previously reported high-resolution information from cryoelectron tomography about the cross-sectional filament bundling architecture. Our study underscores the need for further improvements to the degree of labeling with minimal-offset methods in order to fully exploit molecular-scale optical three-dimensional resolution.

Selective imaging of internalized proteopathic α-synuclein seeds in primary neurons reveals mechanistic insight into transmission of synucleinopathies.

Direct cell-to-cell transmission of proteopathic α-synuclein (α-syn) aggregates is thought to underlie the progression of neurodegenerative synucleinopathies. However, the specific intracellular processes governing this transmission remain unclear because currently available model systems are limited. For example, in cell culture models of α-syn-seeded aggregation, it is difficult to discern intracellular from extracellular exogenously applied α-syn seed species. Herein, we employed fluorescently labeled α-syn preformed fibrils (pffs) in conjunction with the membrane-impermeable fluorescence quencher trypan blue to selectively image internalized α-syn seeds in cultured primary neurons and to quantitatively characterize the concentration dependence, time course, and inhibition of pff uptake. To study the long-term fates of exogenous α-syn pffs in neurons, we developed a pff species labeled at amino acid residue 114 with the environmentally insensitive fluorophore BODIPY or the pH-sensitive dye pHrodo red. We found that pffs are rapidly trafficked along the endolysosomal pathway, where most of the material remains for days. We also found that brief pharmacological perturbation of lysosomes shortly after the pff treatment causes aberrations in intracellular processing of pff seeds concomitant with an increased rate of inclusion formation via recruitment of endogenous α-syn to a relatively small number of exogenous seeds. Our results validate a quantitative assay for pff uptake in primary neurons, implicate lysosomal processing as the major fate of internalized proteopathic seeds, and suggest lysosomal integrity as a significant rate-determining step in the transmission of α-syn pathology. Further, lysosomal processing of transmitted seeds may represent a new therapeutic target to combat the spread of synucleinopathies.

2P-NucTag: on-demand phototagging for molecular analysis of functionally identified cortical neurons.

Neural circuits are characterized by genetically and functionally diverse cell types. A mechanistic understanding of circuit function is predicated on linking the genetic and physiological properties of individual neurons. However, it remains highly challenging to map the functional properties of transcriptionally heterogeneous neuronal subtypes in mammalian cortical circuits in vivo. Here, we introduce a high-throughput two-photon nuclear phototagging (2P-NucTag) approach optimized for on-demand and indelible labeling of single neurons via a photoactivatable red fluorescent protein following in vivo functional characterization in behaving mice. We demonstrate the utility of this function-forward pipeline by selectively labeling and transcriptionally profiling previously inaccessible 'place' and 'silent' cells in the mouse hippocampus. Our results reveal unexpected differences in gene expression between these hippocampal pyramidal neurons with distinct spatial coding properties. Thus, 2P-NucTag opens a new way to uncover the molecular principles that govern the functional organization of neural circuits.

MINSTED fluorescence localization and nanoscopy.

We introduce MINSTED, a fluorophore localization and super-resolution microscopy concept based on stimulated emission depletion (STED) that provides spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED doughnut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background and the required number of fluorescence detections are low compared with most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200-1,000 detections per fluorophore provide a localization precision of 1-3nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a -100-fold improvement in far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells.

Acridonylalanine (Acd) is a fluorescent amino acid that is highly photostable, with a high quantum yield and long fluorescence lifetime in water. These properties make it superior to existing genetically encodable fluorescent amino acids for monitoring protein interactions and conformational changes through fluorescence polarization or lifetime experiments, including fluorescence lifetime imaging microscopy (FLIM). Here, we report the genetic incorporation of Acd using engineered pyrrolysine tRNA synthetase (RS) mutants that allow for efficient Acd incorporation in both E. coli and mammalian cells. We compare protein yields and amino acid specificity for these Acd RSs to identify an optimal construct. We also demonstrate the use of Acd in FLIM, where its long lifetime provides strong contrast compared to endogenous fluorophores and engineered fluorescent proteins, which have lifetimes less than 5 ns.