StayGold variants for molecular fusion and membrane-targeting applications.

Although StayGold is a bright and highly photostable fluorescent protein, its propensity for obligate dimer formation may hinder applications in molecular fusion and membrane targeting. To attain monovalent as well as bright and photostable labeling, we engineered tandem dimers of StayGold to promote dispersibility. On the basis of the crystal structure of this fluorescent protein, we disrupted the dimerization to generate a monomeric variant that offers improved photostability and brightness compared to StayGold. We applied the new monovalent StayGold tools to live-cell imaging experiments using spinning-disk laser-scanning confocal microscopy or structured illumination microscopy. We achieved cell-wide, high-spatiotemporal resolution and sustained imaging of dynamic subcellular events, including the targeting of endogenous condensin I to mitotic chromosomes, the movement of the Golgi apparatus and its membranous derivatives along microtubule networks, the distribution of cortical filamentous actin and the remolding of cristae membranes within mobile mitochondria.

Discovery of a phase-separating small molecule that selectively sequesters tubulin in cells.

Phase-separated membraneless organelles or biomolecular condensates play diverse functions in cells, however recapturing their characteristics using small organic molecules has been a challenge. In the present study, cell-lysate-based screening of 843 self-assembling small molecules led to the discovery of a simple organic molecule, named huezole, that forms liquid droplets to selectively sequester tubulin. Remarkably, this small molecule enters cultured human cells and prevents cell mitosis by forming tubulin-concentrating condensates in cells. The present study demonstrates the feasibility of producing a synthetic condensate out of non-peptidic small molecules for exogenous control of cellular processes. The modular structure of huezole provides a framework for designing a class of organelle-emulating small molecules.

Glucose as a Protein-Condensing Cellular Solute.

The present study reports a surprising protein-condensing effect of glucose, prompted by our accidental observation during chemical library screening under a high-glucose condition. We noticed "glucosing-out" of certain compounds, in which physiological concentrations of glucose induced compound aggregation. Adapting the "glucosing-out" concept to proteins, our proteomic analysis identified three cellular proteins (calmodulin, rho guanine nucleotide exchange factor 40, and polyubiquitin-C) that displayed robust glucose-dependent precipitation. One of these proteins, calmodulin, formed glucose-dependent condensates that control cellular glycogenolysis in hepatic cells. Our findings suggest that glucose is a heretofore underappreciated driver of protein phase separation that may have profound effects on cellular homeostasis.

Extracellular vesicles synchronize cellular phenotypes of differentiating cells.

During embryonic development, cells differentiate in a coordinated manner, aligning their fate decisions and differentiation stages with those of surrounding cells. However, little is known about the mechanisms that regulate this synchrony. Here we show that cells in close proximity synchronize their differentiation stages and cellular phenotypes with each other via extracellular vesicle (EV)-mediated cellular communication. We previously established a mouse embryonic stem cell (ESC) line harbouring an inducible constitutively active protein kinase A (CA-PKA) gene and found that the ESCs rapidly differentiated into mesoderm after PKA activation. In the present study, we performed a co-culture of Control-ESCs and PKA-ESCs, finding that both ESC types rapidly differentiated in synchrony even when PKA was activated only in PKA-ESCs, a phenomenon we named 'Phenotypic Synchrony of Cells (PSyC)'. We further demonstrated PSyC was mediated by EVs containing miR-132. PKA-ESC-derived EVs and miR-132-containing artificial nano-vesicles similarly enhanced mesoderm and cardiomyocyte differentiation in ESCs and ex vivo embryos, respectively. PSyC is a new form of cell-cell communication mediated by the EV regulation of neighbouring cells and could be broadly involved in tissue development and homeostasis.

DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation.

A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1G71A, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.

All-Electrical Ca2+-Independent Signal Transduction Mediates Attractive Sodium Taste in Taste Buds.

Sodium taste regulates salt intake. The amiloride-sensitive epithelial sodium channel (ENaC) is the Na+ sensor in taste cells mediating attraction to sodium salts. However, cells and intracellular signaling underlying sodium taste in taste buds remain long-standing enigmas. Here, we show that a subset of taste cells with ENaC activity fire action potentials in response to ENaC-mediated Na+ influx without changing the intracellular Ca2+ concentration and form a channel synapse with afferent neurons involving the voltage-gated neurotransmitter-release channel composed of calcium homeostasis modulator 1 (CALHM1) and CALHM3 (CALHM1/3). Genetic elimination of ENaC in CALHM1-expressing cells as well as global CALHM3 deletion abolished amiloride-sensitive neural responses and attenuated behavioral attraction to NaCl. Together, sodium taste is mediated by cells expressing ENaC and CALHM1/3, where oral Na+ entry elicits suprathreshold depolarization for action potentials driving voltage-dependent neurotransmission via the channel synapse. Thus, all steps in sodium taste signaling are voltage driven and independent of Ca2+ signals. This work also reveals ENaC-independent salt attraction.

Three-dimensional live imaging of Atoh1 reveals the dynamics of hair cell induction and organization in the developing cochlea.

During cochlear development, hair cells (HCs) and supporting cells differentiate in the prosensory domain to form the organ of Corti, but how one row of inner HCs (IHCs) and three rows of outer HCs (OHCs) are organized is not well understood. Here, we investigated the process of HC induction by monitoring Atoh1 expression in cochlear explants of Atoh1-EGFP knock-in mouse embryos and showed that only the cells that express Atoh1 over a certain threshold are selected for HC fate determination. HC induction initially occurs at the medial edge of the prosensory domain to form IHCs and subsequently at the lateral edge to form OHCs, while Hedgehog signaling maintains a space between IHCs and OHCs, leading to formation of the tunnel of Corti. These results reveal dynamic Atoh1 expression in HC fate control and suggest that multi-directional signals regulate OHC induction, thereby organizing the prototype of the organ of Corti.

Mitochondrial Damage Causes Inflammation via cGAS-STING Signaling in Acute Kidney Injury.

Acute kidney injury (AKI) is characterized by mitochondrial dysfunction and activation of the innate immune system. The cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway detects cytosolic DNA and induces innate immunity. Here, we investigate the role of mitochondrial damage and subsequent activation of the cGAS-STING pathway using a genetically engineered animal model of cisplatin-induced AKI and cultured tubular cells. Cisplatin induced mtDNA leakage into the cytosol-probably through BCL-2-like protein 4 (BAX) pores in the mitochondrial outer membrane-in tubules, with subsequent activation of the cGAS-STING pathway, thereby triggering inflammation and AKI progression, which is improved in STING-deficient mice. STING knockdown in cultured tubular cells ameliorates inflammatory responses induced by cisplatin. mtDNA depletion and repletion studies support tubular inflammatory responses via the cGAS-STING signal activation by cytosolic mtDNA. Therefore, we conclude that mitochondrial dysfunction and subsequent activation of the mtDNA-cGAS-STING pathway is a critical regulator of kidney injury.

Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy.

Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named "actin-pl-clusters". In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent "actin podosome-like clusters".

Leader-Containing Uncapped Viral Transcript Activates RIG-I in Antiviral Stress Granules.

RIG-I triggers antiviral responses by recognizing viral RNA (vRNA) in the cytoplasm. However, the spatio-temporal dynamics of vRNA sensing and signal transduction remain elusive. We investigated the time course of events in cells infected with Newcastle disease virus (NDV), a non-segmented negative-strand RNA virus. RIG-I was recruited to viral replication complexes (vRC) and triggered minimal primary type I interferon (IFN) production. RIG-I subsequently localized to antiviral stress granules (avSG) induced after vRC formation. The inhibition of avSG attenuated secondary IFN production, suggesting avSG as a platform for efficient vRNA detection. avSG selectively captured positive-strand vRNA, and poly(A)+ RNA induced IFN production. Further investigations suggested that uncapped vRNA derived from read-through transcription was sensed by RIG-I in avSG. These results highlight how viral infections stimulate host stress responses, thereby selectively recruiting uncapped vRNA to avSG, in which RIG-I and other components cooperate in an efficient antiviral program.

ScaleS: an optical clearing palette for biological imaging.

Optical clearing methods facilitate deep biological imaging by mitigating light scattering in situ. Multi-scale high-resolution imaging requires preservation of tissue integrity for accurate signal reconstruction. However, existing clearing reagents contain chemical components that could compromise tissue structure, preventing reproducible anatomical and fluorescence signal stability. We developed ScaleS, a sorbitol-based optical clearing method that provides stable tissue preservation for immunochemical labeling and three-dimensional (3D) signal rendering. ScaleS permitted optical reconstructions of aged and diseased brain in Alzheimer's disease models, including mapping of 3D networks of amyloid plaques, neurons and microglia, and multi-scale tracking of single plaques by successive fluorescence and electron microscopy. Human clinical samples from Alzheimer's disease patients analyzed via reversible optical re-sectioning illuminated plaque pathogenesis in the z axis. Comparative benchmarking of contemporary clearing agents showed superior signal and structure preservation by ScaleS. These findings suggest that ScaleS is a simple and reproducible method for accurate visualization of biological tissue.

TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation.

Microtubules (MTs) play critical roles in various cellular events, including cell migration. End-binding proteins (EBs) accumulate at the ends of growing MTs and regulate MT end dynamics by recruiting other plus end-tracking proteins (+TIPs). However, how EBs contribute to MT dynamics through +TIPs remains elusive. We focused on tau-tubulin kinase 2 (TTBK2) as an EB1/3-binding kinase and confirmed that TTBK2 acted as a +TIP. We identified MT-depolymerizing kinesin KIF2A as a novel substrate of TTBK2. TTBK2 phosphorylated KIF2A at S135 in intact cells in an EB1/3-dependent fashion and inactivated its MT-depolymerizing activity in vitro. TTBK2 depletion reduced MT lifetime (facilitated shrinkage and suppressed rescue) and impaired HeLa cell migration, and these phenotypes were partially restored by KIF2A co-depletion. Expression of nonphosphorylatable KIF2A, but not wild-type KIF2A, reduced MT lifetime and slowed down the cell migration. These findings indicate that TTBK2 with EB1/3 phosphorylates KIF2A and antagonizes KIF2A-induced depolymerization at MT plus ends for cell migration.

Real-time imaging of bHLH transcription factors reveals their dynamic control in the multipotency and fate choice of neural stem cells.

The basic-helix-loop-helix (bHLH) transcription factors Ascl1/Mash1, Hes1, and Olig2 regulate the fate choice of neurons, astrocytes, and oligodendrocytes, respectively; however, these factors are coexpressed in self-renewing multipotent neural stem cells (NSCs) even before cell fate determination. This fact raises the possibility that these fate determination factors are differentially expressed between self-renewing and differentiating NSCs with unique expression dynamics. Real-time imaging analysis utilizing fluorescent proteins is a powerful strategy for monitoring expression dynamics. Fusion with fluorescent reporters makes it possible to analyze the dynamic behavior of specific proteins in living cells. However, it is technically challenging to conduct long-term imaging of proteins, particularly those with low expression levels, because a high-sensitivity and low-noise imaging system is required, and very often bleaching of fluorescent proteins and cell toxicity by prolonged laser exposure are problematic. Furthermore, to analyze the functional roles of the dynamic expression of cellular proteins, it is essential to image reporter fusion proteins that are expressed at comparable levels to their endogenous expression. In this review, we introduce our recent reports about the dynamic control of bHLH transcription factors in multipotency and fate choice of NSCs, focusing on real-time imaging of fluorescent reporters fused with bHLH transcription factors. Our imaging results indicate that bHLH transcription factors are expressed in an oscillatory manner by NSCs, and that one of them becomes dominant during fate choice. We propose that the multipotent state of NSCs correlates with the oscillatory expression of several bHLH transcription factors, whereas the differentiated state correlates with the sustained expression of a single bHLH transcription factor.

Mesoscopic metal nanoparticles doubly functionalized with natural and engineered lipidic dispersants for therapeutics.

Surface engineering of mesoscopic metal nanoparticles to increase biocompatibility and cell interaction is important for improvement of their therapeutic properties. Here, we describe a strategy to stabilize mesoscopic metal nanoparticles and to enhance their cell interaction by stepwise addition of (Z)-9-octadecenoate (oleate) and a cell-penetrating peptide-fused high-density lipoprotein (cpHDL). Oleate replaces a cytotoxic dispersant on the surface of gold nanorods (AuNRs), which enables subsequent cpHDL binding without causing aggregation. Notably, these two lipidic dispersants are probably intercalated on the surface. This procedure was also used to stabilize 20 nm spherical gold nanoparticles and 40 nm aggregates of 10 nm magnetite nanoparticles. cpHDL-bound AuNRs were internalized greater than 80 times more efficiently than poly(ethylene glycol)-conjugated AuNRs and were able to elicit cancer cell photoablation.

Plk1 phosphorylates CLIP-170 and regulates its binding to microtubules for chromosome alignment.

The microtubule (MT) cytoskeleton is essential for cellular morphogenesis, cell migration, and cell division. MT organization is primarily mediated by a variety of MT-associated proteins. Among these proteins, plus-end-tracking proteins (+TIPs) are evolutionarily conserved factors that selectively accumulate at growing MT plus ends. Cytoplasmic linker protein (CLIP)-170 is a +TIP that associates with diverse proteins to determine the behavior of MT ends and their linkage to intracellular structures, including mitotic chromosomes. However, how CLIP-170 activity is spatially and temporally controlled is largely unknown. Here, we show that phosphorylation at Ser312 in the third serine-rich region of CLIP-170 is increased during mitosis. Polo-like kinase 1 (Plk1) is responsible for this phosphorylation during the mitotic phase of dividing cells. In vitro analysis using a purified CLIP-170 N-terminal fragment showed that phosphorylation by Plk1 diminishes CLIP-170 binding to the MT ends and lattice without affecting binding to EB3. Furthermore, we demonstrate that during mitosis, stable kinetochore/MT attachment and subsequent chromosome alignment require CLIP-170 and a proper phosphorylation/dephosphorylation cycle at Ser312. We propose that CLIP-170 phosphorylation by Plk1 regulates proper chromosome alignment by modulating the interaction between CLIP-170 and MTs in mitotic cells and that CLIP-170 activity is stringently controlled by its phosphorylation state, which depends on the cellular context.

Oscillatory control of factors determining multipotency and fate in mouse neural progenitors.

The basic helix-loop-helix transcription factors Ascl1/Mash1, Hes1, and Olig2 regulate fate choice of neurons, astrocytes, and oligodendrocytes, respectively. These same factors are coexpressed by neural progenitor cells. Here, we found by time-lapse imaging that these factors are expressed in an oscillatory manner by mouse neural progenitor cells. In each differentiation lineage, one of the factors becomes dominant. We used optogenetics to control expression of Ascl1 and found that, although sustained Ascl1 expression promotes neuronal fate determination, oscillatory Ascl1 expression maintains proliferating neural progenitor cells. Thus, the multipotent state correlates with oscillatory expression of several fate-determination factors, whereas the differentiated state correlates with sustained expression of a single factor.

Rac1 recruitment to the archipelago structure of the focal adhesion through the fluid membrane as revealed by single-molecule analysis.

The focal adhesion (FA) is an integrin-based structure built in/on the plasma membrane (PM), linking the extracellular matrix to the actin stress-fibers, working as cell migration scaffolds. Previously, we proposed the archipelago architecture of the FA, in which FA largely consists of fluid membrane, dotted with small islands accumulating FA proteins: membrane molecules enter the inter-island channels in the FA zone rather freely, and the integrins in the FA-protein islands rapidly exchanges with those in the bulk membrane. Here, we examined how Rac1, a small G-protein regulating FA formation, and its activators αPIX and ßPIX, are recruited to the FA zones. PIX molecules are recruited from the cytoplasm to the FA zones directly. In contrast, majorities of Rac1 molecules first arrive from the cytoplasm on the general inner PM surface, and then enter the FA zones via lateral diffusion on the PM, which is possible due to rapid Rac1 diffusion even within the FA zones, slowed only by a factor of two to four compared with that outside. The constitutively-active Rac1 mutant exhibited temporary and all-time immobilizations in the FA zone, suggesting that upon PIX-induced Rac1 activation at the FA-protein islands, Rac1 tends to be immobilized at the FA-protein islands.

Functional characterization of domains of IPS-1 using an inducible oligomerization system.

The innate immune system recognizes viral nucleic acids and stimulates cellular antiviral responses. Intracellular detection of viral RNA is mediated by the Retinoic acid inducible gene (RIG)-I Like Receptor (RLR), leading to production of type I interferon (IFN) and pro-inflammatory cytokines. Once cells are infected with a virus, RIG-I and MDA5 bind to viral RNA and undergo conformational change to transmit a signal through direct interaction with downstream CARD-containing adaptor protein, IFN-ß promoter stimulator-1 (IPS-1, also referred as MAVS/VISA/Cardif). IPS-1 is composed of N-terminal Caspase Activation and Recruitment Domain (CARD), proline-rich domain, intermediate domain, and C-terminal transmembrane (TM) domain. The TM domain of IPS-1 anchors it to the mitochondrial outer membrane. It has been hypothesized that activated RLR triggers the accumulation of IPS-1, which forms oligomer as a scaffold for downstream signal proteins. However, the exact mechanisms of IPS-1-mediated signaling remain controversial. In this study, to reveal the details of IPS-1 signaling, we used an artificial oligomerization system to induce oligomerization of IPS-1 in cells. Artificial oligomerization of IPS-1 activated antiviral signaling without a viral infection. Using this system, we investigated the domain-requirement of IPS-1 for its signaling. We discovered that artificial oligomerization of IPS-1 could overcome the requirement of CARD and the TM domain. Moreover, from deletion- and point-mutant analyses, the C-terminal Tumor necrosis factor Receptor-Associated Factor (TRAF) binding motif of IPS-1 (aa. 453-460) present in the intermediate domain is critical for downstream signal transduction. Our results suggest that IPS-1 oligomerization is essential for the formation of a multiprotein signaling complex and enables downstream activation of transcription factors, Interferon Regulatory Factor 3 (IRF3) and Nuclear Factor-κB (NF-κB), leading to type I IFN and pro-inflammatory cytokine production.

Full length and delta lactoferrin display differential cell localization dynamics, but do not act as tumor markers or significantly affect the expression of other genes.

Lactoferrin is a secreted protein related to transferrin. Lactoferrin indirectly protects host cells against foreign insults by killing bacteria, scavenging free iron, and binding to receptors required for viral invasion. However, lactoferrin is also proposed to act directly on cells as a transcription factor and tumor suppressor gene. In addition to full length lactoferrin, a truncated form, called delta lactoferrin, can also be produced by alternative splicing. We show here that transformed and nontransformed cells are equally able to express both full length and delta lactoferrin. Moreover, both forms of lactoferrin failed to substantially modulate the expression of other genes. Thus, lactoferrin does not seem to directly control gene expression or inhibit tumor cell growth.