Targeting intracellular oncoproteins with dimeric IgA promotes expulsion from the cytoplasm and immune-mediated control of epithelial cancers.

Dimeric IgA (dIgA) can move through cells via the IgA/IgM polymeric immunoglobulin receptor (PIGR), which is expressed mainly on mucosal epithelia. Here, we studied the ability of dIgA to target commonly mutated cytoplasmic oncodrivers. Mutation-specific dIgA, but not IgG, neutralized KRASG12D within ovarian carcinoma cells and expelled this oncodriver from tumor cells. dIgA binding changed endosomal trafficking of KRASG12D from accumulation in recycling endosomes to aggregation in the early/late endosomes through which dIgA transcytoses. dIgA targeting of KRASG12D abrogated tumor cell proliferation in cell culture assays. In vivo, KRASG12D-specific dIgA1 limited the growth of KRASG12D-mutated ovarian and lung carcinomas in a manner dependent on CD8+ T cells. dIgA specific for IDH1R132H reduced colon cancer growth, demonstrating effective targeting of a cytoplasmic oncodriver not associated with surface receptors. dIgA targeting of KRASG12D restricted tumor growth more effectively than small-molecule KRASG12D inhibitors, supporting the potential of this approach for the treatment of human cancers.

Post-translational modification localizes MYC to the nuclear pore basket to regulate a subset of target genes involved in cellular responses to environmental signals.

The transcription factor MYC (also c-Myc) induces histone modification, chromatin remodeling, and the release of paused RNA polymerase to broadly regulate transcription. MYC is subject to a series of post-translational modifications that affect its stability and oncogenic activity, but how these control MYC's function on the genome is largely unknown. Recent work demonstrates an intimate connection between nuclear compartmentalization and gene regulation. Here, we report that Ser62 phosphorylation and PIN1-mediated isomerization of MYC dynamically regulate the spatial distribution of MYC in the nucleus, promoting its association with the inner basket of the nuclear pore in response to proliferative signals, where it recruits the histone acetyltransferase GCN5 to bind and regulate local gene acetylation and expression. We demonstrate that PIN1-mediated localization of MYC to the nuclear pore regulates MYC target genes responsive to mitogen stimulation that are involved in proliferation and migration pathways. These changes are also present at the chromatin level, with an increase in open regulatory elements in response to stimulation that is PIN1-dependent and associated with MYC chromatin binding. Taken together, our study indicates that post-translational modification of MYC controls its spatial activity to optimally regulate gene expression in response to extrinsic signals in normal and diseased states.

A Versatile Synthetic Pathway for Producing Mesostructured Plasmonic Nanostructures.

Highly branched gold (Au) nanostructures with sharp tips are considered excellent substrates for surface-enhanced Raman scattering (SERS)-based sensing technologies. Here, a simple synthetic route for producing Au or Au-Ag bimetallic mesostructures with multiple sharpened tips in the presence of carbon quantum dots (CQDs) is presented. The morphologies of these mesostructured plasmonic nanoparticles (MSPNs) can be controlled by adjusting the concentration of CQDs, reaction temperatures, and seed particles. The optimal molar ratio for [HAuCl4 ]/[CQDs] is found to be ≈25. At this molar ratio, the diameters of MSPNs can be tuned from 80 to 200 nm by changing the reaction temperature from 25 to 80 °C. In addition, it is found that hierarchical MSPNs consisting of multiple Au nanocrystals can be formed over the entire seed particle surface. Finally, the SERS activity of these MSPNs is examined through the detection of rhodamine 6G and methylene blue. Of the different mesostructures, the bimetallic MSPNs have the highest sensitivity with the ability to detect 10-7  m of rhodamine 6G and 10-6  m of methylene blue. The properties of these MSPN particles, made using a novel synthetic process, make them excellent candidates for SERS-based chemical sensing applications.

The role of coagulation and platelets in colon cancer-associated thrombosis.

Cancer-associated thrombosis is a common first presenting sign of malignancy and is currently the second leading cause of death in cancer patients after their malignancy. However, the molecular mechanisms underlying cancer-associated thrombosis remain undefined. In this study, we aimed to develop a better understanding of how cancer cells affect the coagulation cascade and platelet activation to induce a prothrombotic phenotype. Our results show that colon cancer cells trigger platelet activation in a manner dependent on cancer cell tissue factor (TF) expression, thrombin generation, activation of the protease-activated receptor 4 (PAR4) on platelets and consequent release of ADP and thromboxane A2. Platelet-colon cancer cell interactions potentiated the release of platelet-derived extracellular vesicles (EVs) rather than cancer cell-derived EVs. Our data show that single colon cancer cells were capable of recruiting and activating platelets and generating fibrin in plasma under shear flow. Finally, in a retrospective analysis of colon cancer patients, we found that the number of venous thromboembolism events was 4.5 times higher in colon cancer patients than in a control population. In conclusion, our data suggest that platelet-cancer cell interactions and perhaps platelet procoagulant EVs may contribute to the prothrombotic phenotype of colon cancer patients. Our work may provide rationale for targeting platelet-cancer cell interactions with PAR4 antagonists together with aspirin and/or ADP receptor antagonists as a potential intervention to limit cancer-associated thrombosis, balancing safety with efficacy.

Simultaneous Multicolor Single-Molecule Tracking with Single-Laser Excitation via Spectral Imaging.

Single-molecule tracking (SMT) offers rich information on the dynamics of underlying biological processes, but multicolor SMT has been challenging due to spectral cross talk and a need for multiple laser excitations. Here, we describe a single-molecule spectral imaging approach for live-cell tracking of multiple fluorescent species at once using a single-laser excitation. Fluorescence signals from all the molecules in the field of view are collected using a single objective and split between positional and spectral channels. Images of the same molecule in the two channels are then combined to determine both the location and the identity of the molecule. The single-objective configuration of our approach allows for flexible sample geometry and the use of a live-cell incubation chamber required for live-cell SMT. Despite a lower photon yield, we achieve excellent spatial (20-40 nm) and spectral (10-15 nm) resolutions comparable to those obtained with dual-objective, spectrally resolved Stochastic Optical Reconstruction Microscopy. Furthermore, motions of the fluorescent molecules did not cause loss of spectral resolution owing to the dual-channel spectral calibration. We demonstrate SMT in three (and potentially more) colors using spectrally proximal fluorophores and single-laser excitation, and show that trajectories of each species can be reliably extracted with minimal cross talk.

Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway.

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRas(G12D), a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas(G12D) is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.

Assessment of roles for the Rho-specific guanine nucleotide dissociation inhibitor Ly-GDI in platelet function: a spatial systems approach.

On activation at sites of vascular injury, platelets undergo morphological alterations essential to hemostasis via cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42, and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in platelet function. We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. Whereas RhoGDI localizes throughout platelets in a granule-like manner, Ly-GDI shows an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI, which is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP) VI-specific agonist collagen-related peptide. Additionally, PKC inhibition diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Together, our results suggest a role for Ly-GDI in the localized regulation of Rho GTPases in platelets and hypothesize a link between the PKC and Rho GTPase signaling systems in platelet function.

Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling.

The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF-CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.

Multispectral Localized Surface Plasmon Resonance (msLSPR) Reveals and Overcomes Spectral and Sensing Heterogeneities of Single Gold Nanoparticles.

Metal nanoparticles can be sensitive molecular sensors due to enhanced absorption and scattering of light near a localized surface plasmon resonance (LSPR). Variations in both intrinsic properties such as the geometry and extrinsic properties such as the environment can cause heterogeneity in nanoparticle LSPR and impact the overall sensing responses. To date, however, few studies have examined LSPR and sensing heterogeneities, due to technical challenges in obtaining the full LSPR spectra of individual nanoparticles in dynamic assays. Here, we report multispectral LSPR (msLSPR), a wide-field imaging technique for real-time spectral monitoring of light scattering from individual nanoparticles across the whole field of view (FOV) at ∼0.5 nm spectral and ∼100 ms temporal resolutions. Using msLSPR, we studied the spectral and sensing properties of gold nanoparticles commonly used in LSPR assays, including spheres, rods, and bipyramids. Complemented with electron microscopy imaging, msLSPR analysis revealed that all classes of gold nanoparticles exhibited variations in LSPR peak wavelengths that largely paralleled variations in morphology. Compared with the rods and spheres, gold nanobipyramids exhibited both more uniform and stronger sensing responses as long as the bipyramids are structurally intact. Simulations incorporating the experimental LSPR properties demonstrate the negative impact of spectral heterogeneity on the overall performance of conventional, intensity-based LSPR assays and the ability of msLSPR in overcoming both particle heterogeneity and measurement noise. These results highlight the importance of spectral heterogeneity in LSPR-based sensors and the potential advantage of performing LSPR assays in the spectral domain.

Multiplexed and Millimeter-Scale Fluorescence Nanoscopy of Cells and Tissue Sections via Prism-Illumination and Microfluidics-Enhanced DNA-PAINT.

Fluorescence nanoscopy has become increasingly powerful for biomedical research, but it has historically afforded a small field-of-view (FOV) of around 50 µm × 50 µm at once and more recently up to ∼200 µm × 200 µm. Efforts to further increase the FOV in fluorescence nanoscopy have thus far relied on the use of fabricated waveguide substrates, adding cost and sample constraints to the applications. Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT (PRIME-PAINT) for multiplexed fluorescence nanoscopy across millimeter-scale FOVs. Built upon the well-established prism-type total internal reflection microscopy, PRIME-PAINT achieves robust single-molecule localization with up to ∼520 µm × 520 µm single FOVs and 25-40 nm lateral resolutions. Through stitching, nanoscopic imaging over mm2 sample areas can be completed in as little as 40 min per target. An on-stage microfluidics chamber facilitates probe exchange for multiplexing and enhances image quality, particularly for formalin-fixed paraffin-embedded (FFPE) tissue sections. We demonstrate the utility of PRIME-PAINT by analyzing ∼106 caveolae structures in ∼1,000 cells and imaging entire pancreatic cancer lesions from patient tissue biopsies. By imaging from nanometers to millimeters with multiplexity and broad sample compatibility, PRIME-PAINT will be useful for building multiscale, Google-Earth-like views of biological systems.

A phosphoinositide switch mediates exocyst recruitment to multivesicular endosomes for exosome secretion.

Exosomes are secreted to the extracellular milieu when multivesicular endosomes (MVEs) dock and fuse with the plasma membrane. However, MVEs are also known to fuse with lysosomes for degradation. How MVEs are directed to the plasma membrane for exosome secretion rather than to lysosomes is unclear. Here we report that a conversion of phosphatidylinositol-3-phosphate (PI(3)P) to phosphatidylinositol-4-phosphate (PI(4)P) catalyzed sequentially by Myotubularin 1 (MTM1) and phosphatidylinositol 4-kinase type IIα (PI4KIIα) on the surface of MVEs mediates the recruitment of the exocyst complex. The exocyst then targets the MVEs to the plasma membrane for exosome secretion. We further demonstrate that disrupting PI(4)P generation or exocyst function blocked exosomal secretion of Programmed death-ligand 1 (PD-L1), a key immune checkpoint protein in tumor cells, and led to its accumulation in lysosomes. Together, our study suggests that the PI(3)P to PI(4)P conversion on MVEs and the recruitment of the exocyst direct the exocytic trafficking of MVEs for exosome secretion.

Ras Multimers on the Membrane: Many Ways for a Heart-to-Heart Conversation.

Formation of Ras multimers, including dimers and nanoclusters, has emerged as an exciting, new front of research in the 'old' field of Ras biomedicine. With significant advances made in the past few years, we are beginning to understand the structure of Ras multimers and, albeit preliminary, mechanisms that regulate their formation in vitro and in cells. Here we aim to synthesize the knowledge accrued thus far on Ras multimers, particularly the presence of multiple globular (G-) domain interfaces, and discuss how membrane nanodomain composition and structure would influence Ras multimer formation. We end with some general thoughts on the potential implications of Ras multimers in basic and translational biology.

Multipair Förster Resonance Energy Transfer via Spectrally Resolved Single-Molecule Detection.

Förster resonance energy transfer (FRET) is a powerful tool for studying molecular interactions. Its use for studying interactions involving more than two molecules, however, has been limited by spectral crosstalk among the fluorophores. Here, we report multispectral FRET (msFRET) for imaging multiple pairs of interactions in parallel by spectrally resolving single fluorescent molecules. By using a dual (positional and spectral) channel and wide-field imaging configuration, fluorophores with emission maxima as close as 6-10 nm could be reliably distinguished. We demonstrate msFRET by continuously monitoring the hybridization dynamics among 2 × 2 pairs of DNA oligos in parallel using Cy3 and Cy3.5 as donors and Cy5 and Cy5.5 as acceptors. Aside from studying molecular interactions, msFRET may also find applications in probing fluorophore photophysics during FRET and in multiplexed superresolution imaging.

Fast and Multiplexed Super Resolution Imaging of Fixed and Immunostained Cells with DNA-PAINT-ERS.

Recent advances in super resolution microscopy have enabled imaging at the 10-20 nm scale on a light microscope, providing unprecedented details of native biological structures and processes in intact and hydrated samples. Of the existing strategies, DNA points accumulation in imaging nanoscale topography (DNA-PAINT) affords convenient multiplexing, an important feature in interrogating complex biological systems. A practical limitation of DNA-PAINT, however, has been the slow imaging speed. In its original form, DNA-PAINT imaging of each target takes tens of minutes to hours to complete. To address this challenge, several improved implementations have been introduced. These include DNA-PAINT-ERS (where E = ethylene carbonate; R = repeat sequence; S = spacer), a set of strategies that leads to both accelerated DNA-PAINT imaging speed and improved image quality. With DNA-PAINT-ERS, imaging of typical cellular targets such as microtubules takes only 5-10 min. Importantly, DNA-PAINT-ERS also facilitates multiplexing and can be easily integrated into current workflows for fluorescence staining of biological samples. Here, we provide a detailed, step-by-step guide for fast and multiplexed DNA-PAINT-ERS imaging of fixed and immunostained cells grown on glass substrates as adherent monolayers. The protocol should be readily extended to biological samples of a different format (for example tissue sections) or staining mechanisms (for example using nanobodies). © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of probes for DNA-PAINT-ERS Basic Protocol 2: Sample preparation for imaging membrane targets with DNA-PAINT-ERS in fixed cells Alternate Protocol: Immunostaining of extracted U2OS cells Basic Protocol 3: Super resolution image acquisition and analysis.

Nanoscopic Spatial Association between Ras and Phosphatidylserine on the Cell Membrane Studied with Multicolor Super Resolution Microscopy.

Recent work suggests that Ras small GTPases interact with the anionic lipid phosphatidylserine (PS) in an isoform-specific manner, with direct implications for their biological functions. Studies on PS-Ras associations in cells, however, have relied on immuno-EM imaging of membrane sheets. To study their spatial relationships in intact cells, we have combined the use of Lact-C2-GFP, a biosensor for PS, with multicolor super resolution imaging based on DNA-PAINT. At ~20 nm spatial resolution, the resulting super resolution images clearly show the nonuniform molecular distribution of PS on the cell membrane and its co-enrichment with caveolae, as well as with unidentified membrane structures. Two-color imaging followed by spatial analysis shows that KRas-G12D and HRas-G12V both co-enrich with PS in model U2OS cells, confirming previous observations, yet exhibit clear differences in their association patterns. Whereas HRas-G12V is almost always co-enriched with PS, KRas-G12D is strongly co-enriched with PS in about half of the cells, with the other half exhibiting a more moderate association. In addition, perturbations to the actin cytoskeleton differentially impact PS association with the two Ras isoforms. These results suggest that PS-Ras association is context-dependent and demonstrate the utility of multiplexed super resolution imaging in defining the complex interplay between Ras and the membrane.

An omic and multidimensional spatial atlas from serial biopsies of an evolving metastatic breast cancer.

Mechanisms of therapeutic resistance and vulnerability evolve in metastatic cancers as tumor cells and extrinsic microenvironmental influences change during treatment. To support the development of methods for identifying these mechanisms in individual people, here we present an omic and multidimensional spatial (OMS) atlas generated from four serial biopsies of an individual with metastatic breast cancer during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata that includes treatment times and doses, anatomic imaging, and blood-based response measurements to clinical and exploratory analyses, which includes comprehensive DNA, RNA, and protein profiles; images of multiplexed immunostaining; and 2- and 3-dimensional scanning electron micrographs. These data report aspects of heterogeneity and evolution of the cancer genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples of how integrative analyses of these data reveal potential mechanisms of response and resistance and suggest novel therapeutic vulnerabilities.

Reactive oxygen FIB spin milling enables correlative workflow for 3D super-resolution light microscopy and serial FIB/SEM of cultured cells.

Correlative light and electron microscopy (CLEM) is a powerful tool for defining the ultrastructural context of molecularly-labeled biological specimens, particularly when superresolution fluorescence microscopy (SRM) is used for CLEM. Current CLEM, however, is limited by the stark differences in sample preparation requirements between the two modalities. For CLEM using SRM, the small region of interest (ROI) of either or both modalities also leads to low success rate and imaging throughput. To overcome these limitations, here we present a CLEM workflow based on a novel focused ion beam/scanning electron microscope (FIB/SEM) compatible with common SRM for imaging biological specimen with ultrahigh 3D resolution and improved imaging throughput. By using a reactive oxygen source in a plasma FIB (PFIB) and a rotating sample stage, the novel FIB/SEM was able to achieve several hundreds of micrometer large area 3D analysis of resin embedded cells through a process named oxygen serial spin mill (OSSM). Compared with current FIB mechanisms, OSSM offers gentle erosion, highly consistent slice thickness, reduced charging during SEM imaging, and improved SEM contrast without increasing the dose of post-staining and fixation. These characteristics of OSSM-SEM allowed us to pair it with interferometric photoactivated localization microscopy (iPALM), a recent SRM technique that affords 10-20 nm isotropic spatial resolution on hydrated samples, for 3D CLEM imaging. We demonstrate a CLEM workflow generalizable to using other SRM strategies using mitochondria in human osteosarcoma (U2OS) cells as a model system, where immunostained TOM20, a marker for the mitochondrial outer membrane, was used for iPALM. Owing to the large scan area of OSSM-SEM, it is now possible to select as many FOVs as needed for iPALM and conveniently re-locate them in EM, this improving the imaging throughput. The significantly reduced dose of post-fixation also helped to better preserve the sample ultrastructures as evidenced by the excellent 3D registration between OSSM-SEM and iPALM images and by the accurate localization of TOM20 (by iPALM) to the peripheries of mitochondria (by OSSM-SEM). These advantages make OSSM-SEM an ideal modality for CLEM applications. As OSSM-SEM is still in development, we also discuss some of the remaining issues and the implications to biological imaging with SEM alone or with CLEM.

Subcellular Targeted Nanohoop for One- and Two-Photon Live Cell Imaging.

Fluorophores are powerful tools for interrogating biological systems. Carbon nanotubes (CNTs) have long been attractive materials for biological imaging due to their near-infrared excitation and bright, tunable optical properties. The difficulty in synthesizing and functionalizing these materials with precision, however, has hampered progress in this area. Carbon nanohoops, which are macrocyclic CNT substructures, are carbon nanostructures that possess ideal photophysical characteristics of nanomaterials, while maintaining the precise synthesis of small molecules. However, much work remains to advance the nanohoop class of fluorophores as biological imaging agents. Herein, we report an intracellular targeted nanohoop. This fluorescent nanostructure is noncytotoxic at concentrations up to 50 µM, and cellular uptake investigations indicate internalization through endocytic pathways. Additionally, we employ this nanohoop for two-photon fluorescence imaging, demonstrating a high two-photon absorption cross-section (65 GM) and photostability comparable to a commercial probe. This work further motivates continued investigations into carbon nanohoop photophysics and their biological imaging applications.

Interpretation of cancer mutations using a multiscale map of protein systems.

A major goal of cancer research is to understand how mutations distributed across diverse genes affect common cellular systems, including multiprotein complexes and assemblies. Two challenges­how to comprehensively map such systems and how to identify which are under mutational selection­have hindered this understanding. Accordingly, we created a comprehensive map of cancer protein systems integrating both new and published multi-omic interaction data at multiple scales of analysis. We then developed a unified statistical model that pinpoints 395 specific systems under mutational selection across 13 cancer types. This map, called NeST (Nested Systems in Tumors), incorporates canonical processes and notable discoveries, including a PIK3CA-actomyosin complex that inhibits phosphatidylinositol 3-kinase signaling and recurrent mutations in collagen complexes that promote tumor proliferation. These systems can be used as clinical biomarkers and implicate a total of 548 genes in cancer evolution and progression. This work shows how disparate tumor mutations converge on protein assemblies at different scales.