Charge block-driven liquid-liquid phase separation: A mechanism of how phosphorylation regulates phase behavior of disordered proteins.

Phosphorylation regulates protein function by modulating stereospecific interactions between protein-protein or enzyme-ligand. On the other hand, many bioinformatics studies have demonstrated that phosphorylation preferably occurs in intrinsically disordered regions (IDRs), which do not have any secondary and tertiary structures. Although studies have demonstrated that phosphorylation changes the phase behavior of IDRs, the mechanism, which is distinct from the "stereospecific" effect, had not been elucidated.

Charge block-driven liquid-liquid phase separation - mechanism and biological roles.

Liquid-liquid phase separation (LLPS) has increasingly been found to play pivotal roles in a number of intracellular events and reactions, and has introduced a new paradigm in cell biology to explain protein-protein and enzyme-ligand interactions beyond conventional molecular and biochemical theories. LLPS is driven by the cumulative effects of weak and promiscuous interactions, including electrostatic, hydrophobic and cation-π interactions, among polypeptides containing intrinsically disordered regions (IDRs) and describes the macroscopic behaviours of IDR-containing proteins in an intracellular milieu. Recent studies have revealed that interactions between 'charge blocks' - clusters of like charges along the polypeptide chain - strongly induce LLPS and play fundamental roles in its spatiotemporal regulation.

Yes-associated protein regulates cortical actin architecture and dynamics through intracellular translocation of Rho GTPase-activating protein 18.

Yes-associated protein (YAP) is a transcriptional co-activator that controls the transcription of target genes and modulates the structures of various cytoskeletal architecture as mechanical responses. Although it has been known that YAP regulates actin-regulatory proteins, the detailed molecular mechanism of how they control and coordinate intracellular actin architecture remains elusive. Herein, we aimed to examine the structure and dynamics of intracellular actin architecture from molecular to cellular scales in normal and YAP-knockout (YAP-KO) cells utilizing high-speed atomic force microscopy (HS-AFM) for live-cell imaging and other microscope-based mechanical manipulation and measurement techniques.

Self-assembly of CIP4 drives actin-mediated asymmetric pit-closing in clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is pivotal to signal transduction pathways between the extracellular environment and the intracellular space. Evidence from live-cell imaging and super-resolution microscopy of mammalian cells suggests an asymmetric distribution of actin fibres near the clathrin-coated pit, which induces asymmetric pit-closing rather than radial constriction. However, detailed molecular mechanisms of this 'asymmetricity' remain elusive.

Mechanisms of length-dependent recognition of viral double-stranded RNA by RIG-I.

Retinoic acid-inducible gene I (RIG-I) is the most front-line cytoplasmic viral RNA sensor and induces antiviral immune responses. RIG-I recognizes short double-stranded (dsRNA) (< 500 bp), but not long dsRNA (> 500 bp) to trigger antiviral signaling. Since RIG-I is capable of binding with dsRNA irrespective of size, length-dependent RIG-I signaling remains elusive.

Nuclear import receptors are recruited by FG-nucleoporins to rescue hallmarks of TDP-43 proteinopathy.

Background: Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. Here we study the role of karyopherin-β1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology.

Cell cycle-specific phase separation regulated by protein charge blockiness.

Dynamic morphological changes of intracellular organelles are often regulated by protein phosphorylation or dephosphorylation. Phosphorylation modulates stereospecific interactions among structured proteins, but how it controls molecular interactions among unstructured proteins and regulates their macroscopic behaviours remains unknown. Here we determined the cell cycle-specific behaviour of Ki-67, which localizes to the nucleoli during interphase and relocates to the chromosome periphery during mitosis.

Investigating the morphological dynamics of the plasma membrane by high-speed atomic force microscopy.

Despite numerous recent developments in bioimaging techniques, nanoscale and live-cell imaging of the plasma membrane has been challenging because of the insufficient z-resolution of optical microscopes, as well as the lack of fluorescent probes to specifically label small membrane structures. High-speed atomic force microscopy (HS-AFM) is a powerful tool for visualising the dynamics of a specimen surface and is therefore suitable for observing plasma membrane dynamics. Recent developments in HS-AFM for live-cell imaging have enabled the visualisation of the plasma membrane and the network of cortical actin underneath the membrane in a living cell.

How Do Flaviviruses Hijack Host Cell Functions by Phase Separation?

Viral proteins interact with different sets of host cell components throughout the viral life cycle and are known to localize to the intracellular membraneless organelles (MLOs) of the host cell, where formation/dissolution is regulated by phase separation of intrinsically disordered proteins and regions (IDPs/IDRs). Viral proteins are rich in IDRs, implying that viruses utilize IDRs to regulate phase separation of the host cell organelles and augment replication by commandeering the functions of the organelles and/or sneaking into the organelles to evade the host immune response. This review aims to integrate current knowledge of the structural properties and intracellular localizations of viral IDPs to understand viral strategies in the host cell.

Modulation of actin-binding and -bundling activities of MISP/Caprice by multiple phosphorylation.

The actin cytoskeleton plays critical roles in numerous cellular events and functions, and its spatiotemporal dynamics are maintained and regulated by several actin cofactor proteins. MISP/Caprice is a recently reported actin-bundling protein that is also involved in the progression of mitosis. In this study, we investigated how the actin-regulatory function of MISP is modulated by phosphorylation.

Redox-Sensitive Cysteines Confer Proximal Control of the Molecular Crowding Barrier in the Nuclear Pore.

The nuclear pore complex forms a highly crowded selective barrier with intrinsically disordered regions at the nuclear membrane to coordinate nucleocytoplasmic molecular communications. Although oxidative stress is known to alter the barrier function, the molecular mechanism underlying this adaptive control of the nuclear pore complex remains unknown. Here we uncover a systematic control of the crowding barrier within the nuclear pore in response to various redox environments.

High cell density increases glioblastoma cell viability under glucose deprivation via degradation of the cystine/glutamate transporter xCT (SLC7A11).

The cystine/glutamate transporter system x consists of the light-chain subunit xCT (SLC7A11) and the heavy-chain subunit CD98 (4F2hc or SLC3A2) and exchanges extracellular cystine for intracellular glutamate at the plasma membrane. The imported cystine is reduced to cysteine and used for synthesis of GSH, one of the most important antioxidants in cancer cells. Because cancer cells have increased levels of reactive oxygen species, xCT, responsible for cystine-glutamate exchange, is overexpressed in many cancers, including glioblastoma.

Interactions between non-structured domains of FG- and non-FG-nucleoporins coordinate the ordered assembly of the nuclear pore complex in mitosis.

In this study, we examined how channel-forming subunits of the nuclear pore complex (NPC) are assembled into a selective channel within a highly structured scaffold ring during postmitotic assembly. We focused on non-structured domains of the scaffold Nups and performed in vitro self-assembled particle assays with those derived from channel-forming FG-Nups. We found that non-structured domains of ELYS and Nup35N interacted with channel-forming FG-Nups to form a self-assembled particle.

Mark1 regulates distal airspace expansion through type I pneumocyte flattening in lung development.

During the later stages of lung development, two types of pneumocytes, cuboidal type II (AECII) and flattened type I (AECI) alveolar epithelial cells, form distal lung saccules. Here, we highlight how fibroblasts expressing MAP-microtubule affinity regulating kinase 1 (Mark1) are required for the terminal stages of pulmonary development, called lung sacculation. In Mark1-knockout (KO) mice, distal sacculation and AECI flattening are significantly impaired.

Quantitative proteomics indicate a strong correlation of mitotic phospho-/dephosphorylation with non-structured regions of substrates.

Protein phosphorylation plays a critical role in the regulation and progression of mitosis. >40,000 phosphorylated residues and the associated kinases have been identified to date via proteomic analyses. Although some of these phosphosites are associated with regulation of either protein-protein interactions or the catalytic activity of the substrate protein, the roles of most mitotic phosphosites remain unclear.

Cdk1-mediated DIAPH1 phosphorylation maintains metaphase cortical tension and inactivates the spindle assembly checkpoint at anaphase.

Animal cells undergo rapid rounding during mitosis, ensuring proper chromosome segregation, during which an outward rounding force abruptly increases upon prometaphase entry and is maintained at a constant level during metaphase. Initial cortical tension is generated by the actomyosin system to which both myosin motors and actin network architecture contribute. However, how cortical tension is maintained and its physiological significance remain unknown.

N-terminal dual lipidation-coupled molecular targeting into the primary cilium.

The primary cilium functions as an "antenna" for cell signaling, studded with characteristic transmembrane receptors and soluble protein factors, raised above the cell surface. In contrast to the transmembrane proteins, targeting mechanisms of nontransmembrane ciliary proteins are poorly understood. We focused on a pathogenic mutation that abolishes ciliary localization of retinitis pigmentosa 2 protein and revealed a dual acylation-dependent ciliary targeting pathway.

Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here, we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy (HS-AFM) combined with a confocal laser scanning unit.

Cell type-specific suppression of mechanosensitive genes by audible sound stimulation.

Audible sound is a ubiquitous environmental factor in nature that transmits oscillatory compressional pressure through the substances. To investigate the property of the sound as a mechanical stimulus for cells, an experimental system was set up using 94.0 dB sound which transmits approximately 10 mPa pressure to the cultured cells.

Prolines in the α-helix confer the structural flexibility and functional integrity of importin-β.

The karyopherin family of nuclear transport receptors is composed of a long array of amphiphilic α-helices and undergoes flexible conformational changes to pass through the hydrophobic crowding barrier of the nuclear pore. Here, we focused on the characteristic enrichment of prolines in the middle of the outer α-helices of importin-β. When these prolines were substituted with alanine, nuclear transport activity was reduced drastically and , and caused a severe defect in mitotic progression.