Mosaic-PICASSO: accurate crosstalk removal for multiplex fluorescence imaging.

MOTIVATION: Ultra-multiplexed fluorescence imaging has revolutionized our understanding of biological systems, enabling the simultaneous visualization and quantification of multiple targets within biological specimens. A recent breakthrough in this field is PICASSO, a mutual-information-based technique capable of demixing up to 15 fluorophores without their spectra, thereby significantly simplifying the application of ultra-multiplexed fluorescence imaging. However, this study has identified a limitation of mutual information (MI)-based techniques. They do not differentiate between spatial colocalization and spectral mixing. Consequently, MI-based demixing may incorrectly interpret spatially co-localized targets as non-colocalized, leading to overcorrection. RESULTS: We found that selecting regions within a multiplex image with low-spatial similarity for measuring spectroscopic mixing results in more accurate demixing. This method effectively minimizes overcorrections and promises to accelerate the broader adoption of ultra-multiplex imaging. AVAILABILITY AND IMPLEMENTATION: The codes are available at https://github.com/xing-lab-pitt/mosaic-picasso.

A proposed unified mitotic chromosome architecture.

A molecular architecture is proposed for a representative mitotic chromosome, human chromosome 10. This architecture is built on an interphase chromosome structure based on cryo-electron microscopy (cryo-EM) cellular tomography [J. Sedat et al., Proc. Natl. Acad. Sci. U.S.A., in press], thus unifying chromosome structure throughout the complete mitotic cycle. The basic organizational principle for mitotic chromosomes is specific coiling of the 11-nm nucleosome fiber into large scale, ∼200-nm interphase structures, a Slinky [https://en.wikipedia.org/wiki/Slinky; motif cited in S. Bowerman et al., eLife 10, e65587 (2021)], then further modified with subsequent additional coiling for the final mitotic chromosome structure. The final mitotic chromosome architecture accounts for the dimensional values as well as the well-known cytological configurations. In addition, proof is experimentally provided by digital PCR technology that G1 T cell nuclei are diploid with one DNA molecule per chromosome. Many nucleosome linker DNA sequences, the promotors and enhancers, are suggestive of optimal exposure on the surfaces of the large-scale coils.

A proposed unified interphase nucleus chromosome structure: Preliminary preponderance of evidence.

Cryoelectron tomography of the cell nucleus using scanning transmission electron microscopy and deconvolution processing technology has highlighted a large-scale, 100- to 300-nm interphase chromosome structure, which is present throughout the nucleus. This study further documents and analyzes these chromosome structures. The paper is divided into four parts: 1) evidence (preliminary) for a unified interphase chromosome structure; 2) a proposed unified interphase chromosome architecture; 3) organization as chromosome territories (e.g., fitting the 46 human chromosomes into a 10-µm-diameter nucleus); and 4) structure unification into a polytene chromosome architecture and lampbrush chromosomes. Finally, the paper concludes with a living light microscopy cell study showing that the G1 nucleus contains very similar structures throughout. The main finding is that this chromosome structure appears to coil the 11-nm nucleosome fiber into a defined hollow structure, analogous to a Slinky helical spring [https://en.wikipedia.org/wiki/Slinky; motif used in Bowerman et al., eLife 10, e65587 (2021)]. This Slinky architecture can be used to build chromosome territories, extended to the polytene chromosome structure, as well as to the structure of lampbrush chromosomes.

Molecular organization of mammalian meiotic chromosome axis revealed by expansion STORM microscopy.

During prophase I of meiosis, chromosomes become organized as loop arrays around the proteinaceous chromosome axis. As homologous chromosomes physically pair and recombine, the chromosome axis is integrated into the tripartite synaptonemal complex (SC) as this structure's lateral elements (LEs). While the components of the mammalian chromosome axis/LE-including meiosis-specific cohesin complexes, the axial element proteins SYCP3 and SYCP2, and the HORMA domain proteins HORMAD1 and HORMAD2-are known, the molecular organization of these components within the axis is poorly understood. Here, using expansion microscopy coupled with 2-color stochastic optical reconstruction microscopy (STORM) imaging (ExSTORM), we address these issues in mouse spermatocytes at a resolution of 10 to 20 nm. Our data show that SYCP3 and the SYCP2 C terminus, which are known to form filaments in vitro, form a compact core around which cohesin complexes, HORMADs, and the N terminus of SYCP2 are arrayed. Overall, our study provides a detailed structural view of the meiotic chromosome axis, a key organizational and regulatory component of meiotic chromosomes.

Superresolution imaging reveals nanometer- and micrometer-scale spatial distributions of T-cell receptors in lymph nodes.

T cells become activated when T-cell receptors (TCRs) recognize agonist peptides bound to major histocompatibility complex molecules on antigen-presenting cells. T-cell activation critically relies on the spatiotemporal arrangements of TCRs on the plasma membrane. However, the molecular organizations of TCRs on lymph node-resident T cells have not yet been determined, owing to the diffraction limit of light. Here we visualized nanometer- and micrometer-scale TCR distributions in lymph nodes by light sheet direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM). This dSTORM and SIM approach provides the first evidence, to our knowledge, of multiscale reorganization of TCRs during in vivo immune responses. We observed nanometer-scale plasma membrane domains, known as protein islands, on naïve T cells. These protein islands were enriched within micrometer-sized surface areas that we call territories. In vivo T-cell activation caused the TCR territories to contract, leading to the coalescence of protein islands and formation of stable TCR microclusters.

Ritterostatin GN 1N , a Cephalostatin-Ritterazine Bis-steroidal Pyrazine Hybrid, Selectively Targets GRP78.

Natural products discovered by using agnostic approaches, unlike rationally designed leads or those obtained through high-throughput screening, offer the ability to reveal new biological pathways and, hence, serve as an important vehicle to unveil new avenues in drug discovery. The ritterazine-cephalostatin family of natural products displays robust and potent antitumor activities, with sub-nanomolar growth inhibition against multiple cell lines and potent activity in xenograft models. Herein, we used comparative cellular and molecular biological methods to uncover the ritterazine-cephalostatin cytotoxic mode of action (MOA) in human tumor cells. Our findings indicated that, whereas ritterostatin GN 1N , a cephalostatin-ritterazine hybrid, binds to multiple HSP70s, its cellular trafficking confines activity to the endoplasmic reticulum (ER)-based HSP70 isoform, GRP78. This targeting results in activation of the unfolding protein response (UPR) and subsequent apoptotic cell death.

Probing the electromagnetic field of a 15-nanometre hotspot by single molecule imaging.

When light illuminates a rough metallic surface, hotspots can appear, where the light is concentrated on the nanometre scale, producing an intense electromagnetic field. This phenomenon, called the surface enhancement effect, has a broad range of potential applications, such as the detection of weak chemical signals. Hotspots are believed to be associated with localized electromagnetic modes, caused by the randomness of the surface texture. Probing the electromagnetic field of the hotspots would offer much insight towards uncovering the mechanism generating the enhancement; however, it requires a spatial resolution of 1-2 nm, which has been a long-standing challenge in optics. The resolution of an optical microscope is limited to about half the wavelength of the incident light, approximately 200-300 nm. Although current state-of-the-art techniques, including near-field scanning optical microscopy, electron energy-loss spectroscopy, cathode luminescence imaging and two-photon photoemission imaging have subwavelength resolution, they either introduce a non-negligible amount of perturbation, complicating interpretation of the data, or operate only in a vacuum. As a result, after more than 30 years since the discovery of the surface enhancement effect, how the local field is distributed remains unknown. Here we present a technique that uses Brownian motion of single molecules to probe the local field. It enables two-dimensional imaging of the fluorescence enhancement profile of single hotspots on the surfaces of aluminium thin films and silver nanoparticle clusters, with accuracy down to 1.2 nm. Strong fluorescence enhancements, up to 54 and 136 times respectively, are observed in those two systems. This strong enhancement indicates that the local field, which decays exponentially from the peak of a hotspot, dominates the fluorescence enhancement profile.

Color-Coded Super-Resolution Small-Molecule Imaging.

Although the development of super-resolution microscopy dates back to 1994, its applications have been primarily focused on visualizing cellular structures and targets, including proteins, DNA and sugars. We now report on a system that allows both monitoring of the localization of exogenous small molecules in live cells at low resolution and subsequent super-resolution imaging by using stochastic optical reconstruction microscopy (STORM) on fixed cells. This represents a powerful new tool to understand the dynamics of subcellular trafficking associated with the mode and mechanism of action of exogenous small molecules.

Adding an unnatural covalent bond to proteins through proximity-enhanced bioreactivity.

Natural proteins often rely on the disulfide bond to covalently link side chains. Here we genetically introduce a new type of covalent bond into proteins by enabling an unnatural amino acid to react with a proximal cysteine. We demonstrate the utility of this bond for enabling irreversible binding between an affibody and its protein substrate, capturing peptide-protein interactions in mammalian cells, and improving the photon output of fluorescent proteins.

Single dish gradient screening of small molecule localization.

Understanding trafficking in cells and tissues is one of the most critical steps in exploring the mechanisms and modes of action (MOAs) of a small molecule. Typically, deciphering the role of concentration presents one of the most difficult challenges associated with this task. Herein, we present a practical solution to this problem by developing concentration gradients within single dishes of cells. We demonstrate the method by evaluating fluorescently-labelled probes developed from two classes of natural products that have been identified as potential anti-cancer leads by STORM super-resolution microscopy.

Single-molecule super-resolution light-sheet microscopy.

Single-molecule super-resolution imaging is a new promising tool for investigation of sub-cellular structures. Concurrently, light-sheet microscopy, also known as selective plane illumination microscopy (SPIM), has gained rapid favor with the imaging community in developmental biology due to its fast speed, high contrast, deep penetration, and low phototoxicity. While nearly a dozen reviews thoroughly describe the development of light-sheet microscopy and its technological breakthroughs with a main focus on improving the 3D imaging speed of fish embryos, central nervous system, and other tissues, few have addressed the potential of combining light-sheet microscopy and localization-based super-resolution imaging to achieve sub-diffraction-limited resolution. Adapting light-sheet illumination for single-molecule imaging presents unique challenges for instrumentation and reconstruction algorithms. In this Minireview, we provide an overview of the recent developments that address these challenges. We compare different approaches in super-resolution and light-sheet imaging, address advantages and limitations in each approach, and outline future directions of this emerging field.

Giant suppression of photobleaching for single molecule detection via the Purcell effect.

We report giant suppression of photobleaching and a prolonged lifespan of single fluorescent molecules via the Purcell effect in plasmonic nanostructures. The plasmonic structures enhance the spontaneous emission of excited fluorescent molecules, reduce the probability of activating photochemical reactions that destroy the molecules, and hence suppress the bleaching. Experimentally, we observe up to a 1000-fold increase in the total number of photons that we can harvest from a single fluorescent molecule before it bleaches. This approach demonstrates the potential of using the Purcell effect to manipulate photochemical reactions at the subwavelength scale.

Visualizing the chain-flipping mechanism in fatty-acid biosynthesis.

The acyl carrier protein (ACP) from fatty acid synthases sequesters elongating products within its hydrophobic core, but this dynamic mechanism remains poorly understood. We exploited solvatochromic pantetheine probes attached to ACP that fluoresce when sequestered. The addition of a catalytic partner lures the cargo out of the ACP and into the active site of the enzyme, thus enhancing fluorescence to reveal the elusive chain-flipping mechanism. This activity was confirmed by the use of a dual solvatochromic cross-linking probe and solution-phase NMR spectroscopy. The chain-flipping mechanism was visualized by single-molecule fluorescence techniques, thus demonstrating specificity between the Escherichia coli ACP and its ketoacyl synthase catalytic partner KASII.

Type I Interferon Regulates a Coordinated Gene Network to Enhance Cytotoxic T Cell-Mediated Tumor Killing.

Type I interferons (IFN), which activate many IFN-stimulated genes (ISG), are known to regulate tumorigenesis. However, little is known regarding how various ISGs coordinate with one another in developing antitumor effects. Here, we report that the ISG UBA7 is a tumor suppressor in breast cancer. UBA7 encodes an enzyme that catalyzes the covalent conjugation of the ubiquitin-like protein product of another ISG (ISG15) to cellular proteins in a process known as "ISGylation." ISGylation of other ISGs, including STAT1 and STAT2, synergistically facilitates production of chemokine-receptor ligands to attract cytotoxic T cells. These gene-activation events are further linked to clustering and nuclear relocalization of STAT1/2 within IFN-induced promyelocytic leukemia (PML) bodies. Importantly, this coordinated ISG-ISGylation network plays a central role in suppressing murine breast cancer growth and metastasis, which parallels improved survival in patients with breast cancer. These findings reveal a cooperative IFN-inducible gene network in orchestrating a tumor-suppressive microenvironment. SIGNIFICANCE: We report a highly cooperative ISG network, in which UBA7-mediated ISGylation facilitates clustering of transcription factors and activates an antitumor gene-expression program. These findings provide mechanistic insights into immune evasion in breast cancer associated with UBA7 loss, emphasizing the importance of a functional ISG-ISGylation network in tumor suppression.This article is highlighted in the In This Issue feature, p. 327.

Glioma Stem Cell-Specific Superenhancer Promotes Polyunsaturated Fatty-Acid Synthesis to Support EGFR Signaling.

Glioblastoma ranks among the most aggressive and lethal of all human cancers. Functionally defined glioma stem cells (GSC) contribute to this poor prognosis by driving therapeutic resistance and maintaining cellular heterogeneity. To understand the molecular processes essential for GSC maintenance and tumorigenicity, we interrogated the superenhancer landscapes of primary glioblastoma specimens and in vitro GSCs. GSCs epigenetically upregulated ELOVL2, a key polyunsaturated fatty-acid synthesis enzyme. Targeting ELOVL2 inhibited glioblastoma cell growth and tumor initiation. ELOVL2 depletion altered cellular membrane phospholipid composition, disrupted membrane structural properties, and diminished EGFR signaling through control of fatty-acid elongation. In support of the translational potential of these findings, dual targeting of polyunsaturated fatty-acid synthesis and EGFR signaling had a combinatorial cytotoxic effect on GSCs. SIGNIFICANCE: Glioblastoma remains a devastating disease despite extensive characterization. We profiled epigenomic landscapes of glioblastoma to pinpoint cell state-specific dependencies and therapeutic vulnerabilities. GSCs utilize polyunsaturated fatty-acid synthesis to support membrane architecture, inhibition of which impairs EGFR signaling and GSC proliferation. Combinatorial targeting of these networks represents a promising therapeutic strategy.See related commentary by Affronti and Wellen, p. 1161.This article is highlighted in the In This Issue feature, p. 1143.

Observation of spectral anisotropy of gold nanoparticles.

Metallic nanoparticles synthesized by solution-phase chemistry usually exhibit various polygonal morphologies. The shape is known to have a great impact on a nanoparticle's optical properties, for instance, the surface plasmon resonance frequency. It remains unclear, however, whether the scattering spectrum of nanoparticles is generally anisotropic in the far field as a result. This simple question turns out to be extremely challenging to address because of the particle-to-particle shape inhomogeneity in a bulk sample, and the high sensitivity of surface plasmon resonance to local environments. We report the observation of scattering angle-dependent spectra using a newly developed single-particle tracking spectroscopy (SPS). Furthermore, we show that SPS has provided a way to directly visualize the rotational random walk of individual gold nanoparticles in water for the first time.

Ultrafast two-dimensional infrared vibrational echo chemical exchange experiments and theory.

Ultrafast two-dimensional (2D) infrared vibrational echo experiments and theory are used to examine chemical exchange between solute-solvent complexes and the free solute for the solute phenol and three solvent complex partners, p-xylene, benzene, and bromobenzene, in mixed solvents of the partner and CCl4. The experiments measure the time evolution of the 2D spectra of the hydroxyl (OD) stretching mode of the phenol. The time-dependent 2D spectra are analyzed using time-dependent diagrammatic perturbation theory with a model that includes the chemical exchange (formation and dissociation of the complexes), spectral diffusion of both the complex and the free phenol, orientational relaxation of the complexes and free phenol, and the vibrational lifetimes. The detailed calculations are able to reproduce the experimental results and demonstrate that a method employed previously that used a kinetic model for the volumes of the peaks is adequate to extract the exchange kinetics. The current analysis also yields the spectral diffusion (time evolution of the dynamic line widths) and shows that the spectral diffusion is significantly different for phenol complexes and free phenol.

Adiabatic far-field sub-diffraction imaging.

The limited resolution of a conventional optical imaging system stems from the fact that the fine feature information of an object is carried by evanescent waves, which exponentially decays in space and thus cannot reach the imaging plane. We introduce here an adiabatic lens, which utilizes a geometrically conformal surface to mediate the interference of slowly decompressed electromagnetic waves at far field to form images. The decompression is satisfying an adiabatic condition, and by bridging the gap between far field and near field, it allows far-field optical systems to project an image of the near-field features directly. Using these designs, we demonstrated the magnification can be up to 20 times and it is possible to achieve sub-50 nm imaging resolution in visible. Our approach provides a means to extend the domain of geometrical optics to a deep sub-wavelength scale.

Genetically encoding an electrophilic amino acid for protein stapling and covalent binding to native receptors.

Covalent bonds can be generated within and between proteins by an unnatural amino acid (Uaa) reacting with a natural residue through proximity-enabled bioreactivity. Until now, Uaas have been developed to react mainly with cysteine in proteins. Here we genetically encoded an electrophilic Uaa capable of reacting with histidine and lysine, thereby expanding the diversity of target proteins and the scope of the proximity-enabled protein cross-linking technology. In addition to efficient cross-linking of proteins inter- and intramolecularly, this Uaa permits direct stapling of a protein α-helix in a recombinant manner and covalent binding of native membrane receptors in live cells. The target diversity, recombinant stapling, and covalent targeting of endogenous proteins enabled by this versatile Uaa should prove valuable in developing novel research tools, biological diagnostics, and therapeutics by exploiting covalent protein linkages for specificity, irreversibility, and stability.