Patterning of Oncogenic Ras Clustering in Live Cells Using Vertically Aligned Nanostructure Arrays.

As a dominant oncogenic protein, Ras is well-known to segregate into clusters on the plasma membrane for activating downstream signaling. However, current technologies for direct measurements of Ras clustering are limited to sophisticated high-resolution techniques like electron microscopy and fluorescence lifetime imaging. To further promote fundamental investigations and the related drug development, we hereby introduce a nanobar-based platform which effectively guides Ras clusters into quantifiable patterns in live cells that is resolvable under conventional microscopy. Major Ras isoforms, K-Ras, H-Ras, and N-Ras, were differentiated, as well as their highly prevalent oncogenic mutants G12V and G13D. Moreover, the isoform specificity and the sensitivity of a Ras inhibitor were successfully characterized on nanobars. We envision that this nanobar-based platform will serve as an effective tool to read Ras clustering on the plasma membrane, enabling a novel avenue both to decipher Ras regulations and to facilitate anti-Ras drug development.

Guiding Irregular Nuclear Morphology on Nanopillar Arrays for Malignancy Differentiation in Tumor Cells.

For more than a century, abnormal nuclei in tumor cells, presenting subnuclear invaginations and folds on the nuclear envelope, have been known to be associated with high malignancy and poor prognosis. However, current nuclear morphology analysis focuses on the features of the entire nucleus, overlooking the malignancy-related subnuclear features in nanometer scale. The main technical challenge is to probe such tiny and randomly distributed features inside cells. We here employ nanopillar arrays to guide subnuclear features into ordered patterns, enabling their quantification as a strong indicator of cell malignancy. Both breast and liver cancer cells were validated as well as the quantification of nuclear abnormality heterogeneity. The alterations of subnuclear patterns were also explored as effective readouts for drug treatment. We envision that this nanopillar-enabled quantification of subnuclear abnormal features in tumor cells opens a new angle in characterizing malignant cells and studying the unique nuclear biology in cancer.

A subset of flavaglines inhibits KRAS nanoclustering and activation.

The RAS oncogenes are frequently mutated in human cancers and among the three isoforms (KRAS, HRAS and NRAS), KRAS is the most frequently mutated oncogene. Here, we demonstrate that a subset of flavaglines, a class of natural anti-tumour drugs and chemical ligands of prohibitins, inhibit RAS GTP loading and oncogene activation in cells at nanomolar concentrations. Treatment with rocaglamide, the first discovered flavagline, inhibited the nanoclustering of KRAS, but not HRAS and NRAS, at specific phospholipid-enriched plasma membrane domains. We further demonstrate that plasma membrane-associated prohibitins directly interact with KRAS, phosphatidylserine and phosphatidic acid, and these interactions are disrupted by rocaglamide but not by the structurally related flavagline FL1. Depletion of prohibitin-1 phenocopied the rocaglamide-mediated effects on KRAS activation and stability. We also demonstrate that flavaglines inhibit the oncogenic growth of KRAS-mutated cells and that treatment with rocaglamide reduces non-small-cell lung carcinoma (NSCLC) tumour nodules in autochthonous KRAS-driven mouse models without severe side effects. Our data suggest that it will be promising to further develop flavagline derivatives as specific KRAS inhibitors for clinical applications.

N-Trifluoropropylation of Azoles through N-Vinylation and Sequential Hydrogenation.

Installing a fluoroalkyl group onto the nitrogen atom of azoles represents a potential strategy for lead optimization in medicinal chemistry. Herein, we describe a method for the N-trifluoropropylation of azoles. This process is accomplished using a combination of regioselective N-vinylation and sequential hydrogenation. The two-step sequence is applicable to a diverse set of azoles and tolerates a wide range of functionalities. In addition, we showcase its practicability and utility through the gram-scale synthesis and the late-stage modification of a complex molecule.

Label-Retention Expansion Microscopy (LR-ExM) for Enhanced Fluorescent Signals using Trifunctional Probes.

Expansion microscopy (ExM) is a super-resolution imaging technique that bypasses the diffraction limit of conventional optical microscopy (∼250 nm) by enlarging samples with a swellable hydrogel. Combined with various light microscopes, ExM enables an effective resolution ranging from 5 to 70 nm. ExM has now been successfully applied to cell, tissue, and whole-organism samples, providing biologists with a low-cost strategy to visualize samples at the molecular level. However, fluorescence signal loss easily happens for beginners and with early versions of ExM protocols. Here, we describe a protocol called label-retention expansion microscopy (LR-ExM), which can preserve and enhance the signal of ExM imaging via a series of trifunctional probes. These trifunctional probes are antibody-based and easy to prepare, and thus suit the needs of most laboratories. © 2024 Wiley Periodicals LLC. Basic Protocol: LR-ExM using trifunctional probes for enhanced fluorescent signals.

Coaching ribosome biogenesis from the nuclear periphery.

Severe invagination of the nuclear envelope is a hallmark of cancers, aging, neurodegeneration, and infections. However, the outcomes of nuclear invagination remain unclear. This work identified a new function of nuclear invagination: regulating ribosome biogenesis. With expansion microscopy, we observed frequent physical contact between nuclear invaginations and nucleoli. Surprisingly, the higher the invagination curvature, the more ribosomal RNA and pre-ribosomes are made in the contacted nucleolus. By growing cells on nanopillars that generate nuclear invaginations with desired curvatures, we can increase and decrease ribosome biogenesis. Based on this causation, we repressed the ribosome levels in breast cancer and progeria cells by growing cells on low-curvature nanopillars, indicating that overactivated ribosome biogenesis can be rescued by reshaping nuclei. Mechanistically, high-curvature nuclear invaginations reduce heterochromatin and enrich nuclear pore complexes, which promote ribosome biogenesis. We anticipate that our findings will serve as a foundation for further studies on nuclear deformation.

Expansion microscopy: A chemical approach for super-resolution microscopy.

Super-resolution microscopy is a series of imaging techniques that bypass the diffraction limit of resolution. Since the 1990s, optical approaches, such as single-molecular localization microscopy, have allowed us to visualize biological samples from the sub-organelle to the molecular level. Recently, a chemical approach called expansion microscopy emerged as a new trend in super-resolution microscopy. It physically enlarges cells and tissues, which leads to an increase in the effective resolution of any microscope by the length expansion factor. Compared with optical approaches, expansion microscopy has a lower cost and higher imaging depth but requires a more complex procedure. The integration of expansion microscopy and advanced microscopes significantly pushed forward the boundary of super-resolution microscopy. This review covers the current state of the art in expansion microscopy, including the latest methods and their applications, as well as challenges and opportunities for future research.

Cationic peptides erase memories by removing synaptic AMPA receptors through endophilin-mediated endocytosis.

Administration of the Zeta Inhibitory Peptide (ZIP) interferes with memory maintenance and long-term potentiation (LTP). However, mice lacking its putative target, the protein kinase PKMζ, exhibit normal learning and memory as well as LTP, making ZIP's mechanism unclear. Here, we show that ZIP disrupts LTP by removing surface AMPA receptors through its cationic charge alone. This effect was fully blocked by drugs that block macropinocytosis and is dependent on endophilin A2 (endoA2)-mediated endocytosis. ZIP and other cationic peptides selectively removed newly inserted AMPAR nanoclusters, providing a mechanism by which these peptides erase memories without effects on basal synaptic function. Lastly, cationic peptides can be administered locally and/or systemically and can be combined with local microinjection of macropinocytosis inhibitors to modulate memories on local and brain-wide scales. Our findings have critical implications for an entire field of memory mechanisms and highlight a previously unappreciated mechanism by which memories can be lost.

Label-Retention Expansion Microscopy (LR-ExM) Enables Super-Resolution Imaging and High-Efficiency Labeling.

Expansion microscopy (ExM) is a sample preparation technique that can be combined with most light microscopy methods to increase the resolution. After embedding cells or tissues in swellable hydrogel, samples can be physically expanded three-to sixteen-fold (linear dimension) compared to the original size. Therefore, the effective resolution of any microscope is increased by the expansion factor. A major limitation of the previously introduced ExM is reduced fluorescence after polymerization and the digestion procedure. To overcome this limitation, label-retention expansion microscopy (LR-ExM) has been developed, which prevents signal loss and greatly enhances labeling efficiency using a set of novel trifunctional anchors. This technique allows one to achieve higher resolution when investigating cellular or subcellular structures at a nanometric scale with minimal fluorescent signal loss. LR-ExM can be used not only for immunofluorescence labeling, but also with self-labeling protein tags, such as SNAP- and CLIP-tags, thus achieving higher labeling efficiency. This work presents the procedure and troubleshooting for this immunostaining-based approach, as well as discussion of self-labeling tagging approaches of LR-ExM as an alternative.

Synthesis of CF3-Substituted Alkylamines, 1,2-Bisazoles, and 1,4,5,6-Tetrahydro-1,2,4-triazines from Newly Designed Tetrazole-Activated Trifluoromethyl Alkenes.

A new class of Michael acceptor, tetrazolyl-trifluoromethyl alkenes, has been discovered. They readily undergo Michael-type addition instead of addition-elimination reaction with aliphatic amines and azoles to furnish ß-trifluoromethyl alkylamines and CF3-substituted 1,2-bisazole derivatives, respectively. Additionally, some of the products are capable of engaging in microwave-assisted intramolecular denitrogenative annulation, leading to the formation of CF3-substituted 1,4,5,6-tetrahydro-1,2,4-triazines that are otherwise difficult to access by other methodologies.

Comparative Study of Curvature Sensing Mediated by F-BAR and an Intrinsically Disordered Region of FBP17.

Membrane curvature has emerged as an intriguing physical principle underlying biological signaling and membrane trafficking. The CIP4/FBP17/Toca-1 F-BAR subfamily is unique in the BAR family because its structurally folded F-BAR domain does not contain any hydrophobic motifs that insert into membrane. Although widely assumed so, whether the banana-shaped F-BAR domain alone can sense curvature has never been experimentally demonstrated. Using a nanobar-supported lipid bilayer system, we found that the F-BAR domain of FBP17 displayed minimal curvature sensing in vitro. In comparison, an alternatively spliced intrinsically disordered region (IDR) adjacent to the F-BAR domain has the membrane curvature-sensing ability greatly exceeding that of F-BAR domain alone. In living cells, the presence of the IDR delayed the recruitment of FBP17 in curvature-coupled cortical waves. Collectively, we propose that contrary to the common belief, FBP17's curvature-sensing capability largely originates from IDR, and not the F-BAR domain alone.

Membrane curvature sensing of the lipid-anchored K-Ras small GTPase.

Plasma membrane (PM) curvature defines cell shape and intracellular organelle morphologies and is a fundamental cell property. Growth/proliferation is more stimulated in flatter cells than the same cells in elongated shapes. PM-anchored K-Ras small GTPase regulates cell growth/proliferation and plays key roles in cancer. The lipid-anchored K-Ras form nanoclusters selectively enriched with specific phospholipids, such as phosphatidylserine (PS), for efficient effector recruitment and activation. K-Ras function may, thus, be sensitive to changing lipid distribution at membranes with different curvatures. Here, we used complementary methods to manipulate membrane curvature of intact/live cells, native PM blebs, and synthetic liposomes. We show that the spatiotemporal organization and signaling of an oncogenic mutant K-Ras G12V favor flatter membranes with low curvature. Our findings are consistent with the more stimulated growth/proliferation in flatter cells. Depletion of endogenous PS abolishes K-Ras G12V PM curvature sensing. In cells and synthetic bilayers, only mixed-chain PS species, but not other PS species tested, mediate K-Ras G12V membrane curvature sensing. Thus, K-Ras nanoclusters act as relay stations to convert mechanical perturbations to mitogenic signaling.

Biosensor-Based Evolution and Elucidation of a Biosynthetic Pathway in Escherichia coli.

The successful evolution of metabolite-producing microbes requires a high-throughput screening method to obtain the desired properties within a short time. In this study, we developed a transcription-factor-driven device that combines a metabolite-responsive element and a selection module. This device was able to specifically sense intracellular l-phenylalanine (l-Phe) and convert this signal into an observable phenotype. Applying this device, we successfully improved l-Phe production by screening hyperproducing phenotypes from a ribonucleotide binding site library and a random mutagenesis library. In addition, several site mutations introduced by random mutagenesis were identified and elucidated to facilitate the improvement of l-Phe production. Our results present a paradigm for screening of compounds that are not easily observable to raise the yield of targeted compounds from a large candidate library. This approach may guide further applications in rewiring metabolic circuits and facilitate the directed evolution of recombinant strains.