ORP9-PH domain-based fluorescent reporters for visualizing phosphatidylinositol 4-phosphate dynamics in living cells.

Fluorescent reporters that visualize phosphatidylinositol 4-phosphate (PI4P) in living cells are indispensable to elucidate the roles of this fundamental lipid in cell physiology. However, currently available PI4P reporters have limitations, such as Golgi-biased localization and low detection sensitivity. Here, we present a series of fluorescent PI4P reporters based on the pleckstrin homology (PH) domain of oxysterol-binding protein-related protein 9 (ORP9). We show that the green fluorescent protein AcGFP1-tagged ORP9-PH domain can be used as a fluorescent PI4P reporter to detect cellular PI4P across its wide distribution at multiple cellular locations, including the plasma membrane (PM), Golgi, endosomes, and lysosomes with high specificity and contrast. We also developed blue, red, and near-infrared fluorescent PI4P reporters suitable for multicolor fluorescence imaging experiments. Finally, we demonstrate the utility of the ORP9-PH domain-based reporter to visualize dynamic changes in the PI4P distribution and level in living cells upon synthetic ER-PM membrane contact manipulation and GPCR stimulation. This work offers a new set of genetically encoded fluorescent PI4P reporters that are practically useful for the study of PI4P biology.

Palmitoylation-Dependent Small-Molecule Fluorescent Probes for Live-Cell Golgi Imaging.

Small-molecule fluorescent probes enabling visualization of the Golgi apparatus in living cells are essential tools for studying Golgi-associated biological processes and diseases. So far, several fluorescent Golgi stains have been developed by linking ceramide lipids to fluorophores. However, ceramide-based probes suffer from cumbersome staining procedures and low Golgi specificity. Here, we introduce fluorescent Golgi-staining probes based on the tri-N-methylated myristoyl-Gly-Cys (myrGC3Me) motif. The cell-permeable myrGC3Me motif localizes to the Golgi membrane upon S-palmitoylation. By modularly conjugating the myrGC3Me motif to fluorophores, we developed blue, green, and red fluorescent Golgi probes, all of which allowed simple and rapid staining of the Golgi in living cells with high specificity and no cytotoxicity. The probe was also applicable to the visualization of dynamic changes of the Golgi morphology induced by drug treatments and during cell division. The present work provides an entirely new series of live-cell Golgi probes useful for cell biological and diagnostic applications.

Miniaturized Synthetic Palmitoylation Motifs for Small-Molecule Localization in Living Cells.

Conjugating small-molecule ligands to synthetic motifs that can localize to specific organelles or membranes in living cells is a practical approach to develop compounds as chimeric tools or drugs that can manipulate biological processes in a subcellular site-specific manner. However, the number of available organelle-targeted synthetic motifs for small-molecule localization is limited. We have recently developed a synthetic myristoyl-DCys motif for small-molecule localization that undergoes S-palmitoylation via the cellular palmitoylation machinery and localizes to the Golgi surface. Herein, we show that the lipid acyl chain of the myristoyl (C14)-DCys motif can be as short as 10-carbons and still retain the palmitoylation-dependent Golgi localization property in cells. This discovery led to the identification of four new derivatives for small-molecule localization: tridecanoyl (C13)-, dodecanoyl (C12)-, undecanoyl (C11)-, and decanoyl (C10)-DCys motifs. We demonstrated that even the short decanoyl-DCys palmitoylation motif could be used to generate small-molecule ligand conjugates that functioned as chemical tools for controlling protein localization and cell signaling. The miniaturized synthetic palmitoylation motifs identified in this work may find applications in creating various Golgi-localizable chimeric molecules for use in chemical biology and drug development.

A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation.

Chemogenetic methods enabling the rapid translocation of specific proteins to the plasma membrane (PM) in a single protein-single ligand manner are useful tools in cell biology. We recently developed a technique, in which proteins fused to an Escherichia coli dihydrofolate reductase (eDHFR) variant carrying N-terminal hexalysine residues are recruited from the cytoplasm to the PM using the synthetic myristoyl-d-Cys-tethered trimethoprim (mDcTMP) ligand. However, this system achieved PM-specific translocation only when the eDHFR tag was fused to the N terminus of proteins, thereby limiting its application. In this report, we engineered a universal PM-targeting tag for mDcTMP-induced protein translocation by grafting the hexalysine motif into an intra-loop region of eDHFR. We demonstrate the broad applicability of the new loop-engineered eDHFR tag and mDcTMP pair for conditional PM recruitment and activation of various tag-fused signaling proteins with different fusion configurations and for reversibly and repeatedly controlling protein localization to generate synthetic signal oscillations.

A Peptide Nanocage Constructed by Self-Assembly of Oligoproline Conjugates.

Cage-like supramolecular assemblies called molecular cages, which possess attractive functions, have been prepared. Although biomolecule-based nanocages are required for biological/medical applications such as drug delivery systems, the majority of nanocages are constructed using aromatic compounds with lower biocompatibility and biodegradability. In this study, the construction of a peptide nanocage consisting of an oligoproline conjugate is demonstrated. The conjugate was easy to prepare and had high biocompatibility. The oligoproline moiety of the conjugate had a rigid, rod-like structure suitable for the backbone of the supramolecular nanocage. The conjugates self-assembled to form peptide nanocages with a huge inner cavity.

Synthetic Protein Condensates That Inducibly Recruit and Release Protein Activity in Living Cells.

Compartmentation of proteins into biomolecular condensates or membraneless organelles formed by phase separation is an emerging principle for the regulation of cellular processes. Creating synthetic condensates that accommodate specific intracellular proteins on demand would have various applications in chemical biology, cell engineering, and synthetic biology. Here, we report the construction of synthetic protein condensates capable of recruiting and/or releasing proteins of interest in living mammalian cells in response to a small molecule or light. By a modular combination of a tandem fusion of two oligomeric proteins, which forms phase-separated synthetic protein condensates in cells, with a chemically induced dimerization tool, we first created a chemogenetic protein condensate system that can rapidly recruit target proteins from the cytoplasm to the condensates by addition of a small-molecule dimerizer. We next coupled the protein-recruiting condensate system with an engineered proximity-dependent protease, which gave a second protein condensate system wherein target proteins previously expressed inside the condensates are released into the cytoplasm by small-molecule-triggered protease recruitment. Furthermore, an optogenetic condensate system that allows reversible release and sequestration of protein activity in a repeatable manner using light was constructed successfully. These condensate systems were applicable to control protein activity and cellular processes such as membrane ruffling and ERK signaling in a time scale of minutes. This proof-of-principle work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step toward tailor-made engineering of synthetic protein condensate-based soft materials with various functionalities for biological and biomedical applications.

Human Ribosomal RNA-Derived Resident MicroRNAs as the Transmitter of Information upon the Cytoplasmic Cancer Stress.

Dysfunction of ribosome biogenesis induces divergent ribosome-related diseases including ribosomopathy and occasionally results in carcinogenesis. Although many defects in ribosome-related genes have been investigated, little is known about contribution of ribosomal RNA (rRNA) in ribosome-related disorders. Meanwhile, microRNA (miRNA), an important regulator of gene expression, is derived from both coding and noncoding region of the genome and is implicated in various diseases. Therefore, we performed in silico analyses using M-fold, TargetScan, GeneCoDia3, and so forth to investigate RNA relationships between rRNA and miRNA against cellular stresses. We have previously shown that miRNA synergism is significantly correlated with disease and the miRNA package is implicated in memory for diseases; therefore, quantum Dynamic Nexus Score (DNS) was also calculated using MESer program. As a result, seventeen RNA sequences identical with known miRNAs were detected in the human rRNA and termed as rRNA-hosted miRNA analogs (rmiRNAs). Eleven of them were predicted to form stem-loop structures as pre-miRNAs, and especially one stem-loop was completely identical with hsa-pre-miR-3678 located in the non-rDNA region. Thus, these rmiRNAs showed significantly high DNS values, participation in regulation of cancer-related pathways, and interaction with nucleolar RNAs, suggesting that rmiRNAs may be stress-responsible resident miRNAs which transmit stress-tuning information in multiple levels.