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. A series of mutation studies demonstrated that phosphorylation of S394, S395, and S400 induced stress fiber formation in interphase cells. In vitro studies revealed that these phosphorylation events increased the actin-bundling activity but not the actin-binding activity of MISP. Moreover, actin-binding activity was suppressed by mitotic phosphorylation, including that at S376, S471, and S541. These results indicate that phosphorylation during interphase and mitosis differentially regulates the actin-binding and -bundling activities of MISP, in turn regulating the higher-order architecture of the actin cytoskeleton during cell cycle.

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

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-ß. When these prolines were substituted with alanine, nuclear transport activity was reduced drastically in vivo and in vitro, and caused a severe defect in mitotic progression. These mutations did not alter the overall folding of the helical repeat or affect its interaction with cargo or the regulatory factor Ran. However, in vitro and in silico analyses revealed that the mutant lost structural flexibility and could not undergo rapid conformational changes when transferring from a hydrophilic to hydrophobic environment or vice versa. These findings reveal the essential roles of prolines in ensuring the structural flexibility and functional integrity of karyopherins.

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. Short N-terminal sequences which contain myristoylation and palmitoylation sites are sufficient to target a marker protein into the cilium in a palmitoylation-dependent manner. A Golgi-localized palmitoyltransferase DHHC-21 was identified as the key enzyme controlling this targeting pathway. Rapid turnover of the targeted protein was ensured by cholesterol-dependent membrane fluidity, which balances highly and less-mobile populations of the molecules within the cilium. This targeting signal was found in a set of signal transduction molecules, suggesting a general role of this pathway in proper ciliary organization, and dysfunction in ciliary disorders.

Probing in vivo dynamics of mitochondria and cortical actin networks using high-speed atomic force/fluorescence microscopy.

The dynamics of the cell membrane and submembrane structures are closely linked, facilitating various cellular activities. Although cell surface research and cortical actin studies have shown independent mechanisms for the cell membrane and the actin network, it has been difficult to obtain a comprehensive understanding of the dynamics of these structures in live cells. Here, we used a combined atomic force/optical microscope system to analyze membrane-based cellular events at nanometer-scale resolution in live cells. Imaging the COS-7 cell surface showed detailed structural properties of membrane invagination events corresponding to endocytosis and exocytosis. In addition, the movement of mitochondria and the spatiotemporal dynamics of the cortical F-actin network were directly visualized in vivo. Cortical actin microdomains with sizes ranging from 1.7×10(4) to 1.4×10(5) nm2 were dynamically rearranged by newly appearing actin filaments, which sometimes accompanied membrane invaginations, suggesting that these events are integrated with the dynamic regulation of submembrane organizations maintained by actin turnovers. These results provide novel insights into the structural aspects of the entire cell membrane machinery which can be visualized with high temporal and spatial resolution.

Intermolecular disulfide bonds between nucleoporins regulate karyopherin-dependent nuclear transport.

Disulfide (S-S) bonds play important roles in the regulation of protein function and cellular stress responses. In this study, we demonstrate that distinct sets of nucleoporins (Nups), components of the nuclear pore complex (NPC), form S-S bonds and regulate nuclear transport through the NPC. Kinetic analysis of importin ß demonstrated that the permeability of the NPC was increased by dithiothreitol treatment and reduced by oxidative stress. The permeability of small proteins such as GFP was not affected by either oxidative stress or a reducing reagent. Immunoblot analysis revealed that the oxidative stress significantly induced S-S bond formation in Nups 358, 155, 153 and 62 but not 88 and 160. The direct involvement of cysteine residues in the formation of S-S bonds was confirmed by mutating conserved cysteine residues in Nup62, which abolished the formation of S-S bonds and enhanced the permeability of the NPC. Knocking down Nup62 reduced the stress-inducible S-S bonds of Nup155, suggesting that Nup62 and Nup155 are covalently coupled via S-S bonds. From these results, we propose that the inner channel of the NPC is somehow insulated from the cytoplasm and is more sensitive than the cytoplasm to the intracellular redox state.

Caprice/MISP is a novel F-actin bundling protein critical for actin-based cytoskeletal reorganizations.

Caprice [C19orf21 actin-bundling protein in characteristic epithelial cells, also called mitotic interactor and substrate of Plk1 (MISP)] is a novel actin-related protein identified in the highly-insoluble subcellular scaffold proteins. This protein contains multiple actin-binding sites, forms characteristic mesh-like F-actin bundles in vitro, and exhibits capricious localization and expression patterns in vivo. Overexpression or knock-down of Caprice resulted in a dramatic effect on cellular morphology by inducing stress fiber-like thick filaments or filopodial formations, respectively. Caprice is expressed and localized in distinct cells and tissues with specialized actin-based structures, such as growth cones of migrating neurons and stereocilia of inner ear hair cells. However, Caprice gene expression is varied among different cell types; especially enriched in several epithelial cells whereas relatively suppressed in a subset of epithelial cells, fibroblasts, and neuroblastoma cells at the transcriptional level. Thus, this protein is expected to be an effector for cell type-specific actin reorganization with its direct actin-binding properties and provides a novel model of cell morphology regulation by a non-ubiquitous single actin-bundling protein.

Dynamics of WD-repeat containing proteins in SSU processome components.

Nine WD-repeat containing proteins in human SSU processome components have been found in a HeLa cell nuclear matrix fraction. In these proteins, t-UTP sub-complex components, i.e., CIRH1A, UTP15, and WDR43, were shown to be immobilized in the fibrillar centers of nucleoli in living cells. In this study, the dynamics of the remaining six proteins fused with green fluorescent protein (GFP), i.e., PWP2-GFP, TBL3-GFP, GFP-UTP18, GFP-NOL10, GFP-WDR46, and GFP-WDSOF1, were examined in living cells. The findings were as follows. (i) The majority of UTP-B sub-complex components, i.e., PWP2-GFP, TBL3-GFP, and GFP-UTP18, are localized to the dense fibrillar component and granular component regions in nucleoli; (ii) When rRNA transcription is suppressed, the majority of GFP-fused UTP-B sub-complex components are localized in the cap and body regions of nucleoli. (iii) The mobility of these proteins except for GFP-WDSOF1, and half of GFP-UTP18 and GFP-WDR46, respectively, is very low in living cells. (iv) When rRNA transcription is suppressed, the mobility of these proteins except for GFP-WDSOF1 is accelerated but still slow. These findings and others suggest that these WD-repeat proteins other than GFP-WDSOF1 found in the nuclear matrix fraction bind tightly to some macro-protein complexes and act as a scaffold or a core for the complexes in nucleoli.

Karyopherin-independent spontaneous transport of amphiphilic proteins through the nuclear pore.

Highly selective nucleocytoplasmic molecular transport is critical to eukaryotic cells, which is illustrated by size-filtering diffusion and karyopherin-mediated passage mechanisms. However, a considerable number of large proteins without nuclear localization signals are localized to the nucleus. In this paper, we provide evidence for the spontaneous migration of large proteins in a karyopherin-independent manner. Time-lapse observation of a nuclear transport assay revealed that several large molecules spontaneously and independently pass through the nuclear pore complex (NPC). The amphiphilic motifs were sufficient to overcome the selectivity barrier of the NPC. Furthermore, the amphiphilic property of these proteins enables altered local conformation in hydrophobic solutions so that elevated surface hydrophobicity facilitates passage through the nuclear pore. The molecular dynamics simulation revealed the conformational change of the amphiphilic structure that exposes the hydrophobic amino acid residues to the outer surface in a hydrophobic solution. These results contribute to the understanding of nucleocytoplasmic molecular sorting and the nature of the permeability barrier.

Nucleolar scaffold protein, WDR46, determines the granular compartmental localization of nucleolin and DDX21.

The nuclear scaffold is an insoluble nuclear structure that contributes to the inner nuclear organization. In this study, we showed that one of the nuclear scaffold proteins, WDR46, plays a role as a fundamental scaffold component of the nucleolar structure. WDR46 is a highly insoluble nucleolar protein, and its subcellular localization is dependent on neither DNA nor RNA. The N- and C-terminal regions of WDR46 are predicted to be intrinsically disordered, and both regions are critical for the nucleolar localization of WDR46 and the association with its binding partners. When WDR46 was knocked down, two of its binding partners, nucleolin and DDX21 (involved in 18S rRNA processing), were mislocalized from the granular component to the edges of the nucleoli, whereas other binding partners, NOP2 and EBP2 (involved in 28S rRNA processing), were not affected. This is because the proper recruitment of nucleolin and DDX21 to the nucleoli in daughter cells after cell division is ensured by WDR46. These findings suggest a structural role for WDR46 in organizing the 18S ribosomal RNA processing machinery. This role of WDR46 is enabled by its interaction property via intrinsically disordered regions.

Probing the stiffness of isolated nucleoli by atomic force microscopy.

In eukaryotic cells, ribosome biogenesis occurs in the nucleolus, a membraneless nuclear compartment. Noticeably, the nucleolus is also involved in several nuclear functions, such as cell cycle regulation, non-ribosomal ribonucleoprotein complex assembly, aggresome formation and some virus assembly. The most intriguing question about the nucleolus is how such dynamics processes can occur in such a compact compartment. We hypothesized that its structure may be rather flexible. To investigate this, we used atomic force microscopy (AFM) on isolated nucleoli. Surface topography imaging revealed the beaded structure of the nucleolar surface. With the AFM's ability to measure forces, we were able to determine the stiffness of isolated nucleoli. We could establish that the nucleolar stiffness varies upon drastic morphological changes induced by transcription and proteasome inhibition. Furthermore, upon ribosomal proteins and LaminB1 knockdowns, the nucleolar stiffness was increased. This led us to propose a model where the nucleolus has steady-state stiffness dependent on ribosome biogenesis activity and requires LaminB1 for its flexibility.

Antibody-based analysis reveals "filamentous vs. non-filamentous" and "cytoplasmic vs. nuclear" crosstalk of cytoskeletal proteins.

To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do not take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus.

Interaction, mobility, and phosphorylation of human orthologues of WD repeat-containing components of the yeast SSU processome t-UTP sub-complex.

We previously proposed a dynamic scaffold model for inner nuclear structure formation. In this model, structures in inter-chromatin regions are maintained through dynamic interaction of protein complex modules, and WD repeat- and disordered region-rich proteins and others act as scaffolds for these protein complexes. In this study, three WD-repeat proteins, i.e., CIRH1A, UTP15, and WDR43, were found in the nuclear matrix fraction and speculated to be present in the human t-UTP sub-complex of SSU processomes. The results obtained as to their subnuclear localization, binding with each other, mobilities, and phosphorylation were: (i) the majority of these proteins fused with GFP are localized to the fibrillar center region in nucleoli. (ii) these 3 proteins bind directly with each other in vitro. (iii) the movement of these proteins is very slow in living cells and independent of rDNA transcription. (iv) His-CIRH1A is phosphorylated at Thr(131) by a mitotic Xenopus egg extract, and binding with GST-UTP15 and GST-WDR43 is suppressed. These findings and others suggest that these 3 WD proteins found in the matrix fraction bind directly with each other, bind tightly to fibrillar center regions, and comprise a part of the nucleolar structure. These results are also consistent with our dynamic scaffold model.

Molecular mechanisms underlying nucleocytoplasmic shuttling of actinin-4.

In addition to its well-known role as a crosslinker of actin filaments at focal-adhesion sites, actinin-4 is known to be localized to the nucleus. In this study, we reveal the molecular mechanism underlying nuclear localization of actinin-4 and its novel interactions with transcriptional regulators. We found that actinin-4 is imported into the nucleus through the nuclear pore complex in an importin-independent manner and is exported by the chromosome region maintenance-1 (CRM1)-dependent pathway. Nuclear actinin-4 levels were significantly increased in the late G2 phase of the cell cycle and were decreased in the G1 phase, suggesting that active release from the actin cytoskeleton was responsible for increased nuclear actinin-4 in late G2. Nuclear actinin-4 was found to interact with the INO80 chromatin-remodeling complex. It also directs the expression of a subset of cell-cycle-related genes and interacts with the upstream-binding factor (UBF)-dependent rRNA transcriptional machinery in the M phase. These findings provide molecular mechanisms for both nucleocytoplasmic shuttling of proteins that do not contain a nuclear-localization signal and cell-cycle-dependent gene regulation that reflects morphological changes in the cytoskeleton.

Evolutionary dynamics of spliceosomal intron revealed by in silico analyses of the P-Type ATPase superfamily genes.

It has been long debated whether spliceosomal introns originated in the common ancestor of eukaryotes and prokaryotes. In this study, we tested the possibility that extant introns were inherited from the common ancestor of eukaryotes and prokaryotes using in silico simulation. We first identified 21 intron positions that are shared among different families of the P-Type ATPase superfamily, some of which are known to have diverged before the separation of prokaryotes and eukaryotes. Theoretical estimates of the expected number of intron positions shared by different genes suggest that the introns at those 21 positions were inserted independently. There seems to be no intron that arose from before the diversification of the P-Type ATPase superfamily. Namely, the present introns were inserted after the separation of eukaryotes and prokaryotes.

Ultrasonic control of neurite outgrowth direction.

This study investigated a method to control neurite outgrowth direction using ultrasound vibration. An ultrasound cell culture dish comprising a glass-bottom culture surface and a glass disc with an ultrasound transducer was fabricated, and undifferentiated neuron-like PC12 cells were grown on the dish as an adherent culture. The 78 kHz resonant concentric flexural vibration mode of the dish was used to quantitatively evaluate the neurite outgrowth direction and length. Time-lapse imaging of cells was performed for 72 h under ultrasound excitation. Unsonicated neurites grew in random directions, whereas neurite outgrowth was circumferentially oriented during ultrasonication in a power-dependent manner. The neurite orientation correlated with the spatial gradient of the ultrasound vibration, implying that neurites tend to grow in directions along which the vibrational amplitude does not change. Ultrasonication with 30 Vpp for 72 h increased the neurite length by 99.7% compared with that observed in unsonicated cells.

Proteomic and targeted analytical identification of BXDC1 and EBNA1BP2 as dynamic scaffold proteins in the nucleolus.

The nuclear matrix has classically been assumed to be a solid structure coherently aligning nuclear components, but its real nature remains obscure. We separated the proteins in a ribonucleoprotein-containing nuclear matrix fraction of HeLa cells by reversed-phase HPLC followed by SDS-PAGE, and identified 83 proteins through peptide mass fingerprint (PMF) analysis. Many nucleolar proteins, classical nuclear matrix proteins, RNA binding proteins, cytoskeletal proteins and five uncharacterized proteins were identified in this fraction. Four of the latter proteins were localized to the cell nucleus, BXDC1 and EBNA1BP2 being especially localized to the nucleolus. Fluorescence recovery after photobleaching and RNAi knockdown analyses suggested that BXDC1 and EBNA1BP2 function in a dynamic scaffold for ribosome biogenesis.

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. Direct measurements of the crowding states using a crowding-sensitive FRET (Förster resonance energy transfer) probe reveal specific roles of the nuclear pore subunits that adjust the degree of crowding in response to different redox conditions, by adaptively forming or disrupting redox-sensitive disulfide bonds. Relationships between crowding control and the barrier function of the nuclear pore are investigated by single-molecular fluorescence measurements of nuclear transport. Based on these findings, we propose a proximal control model of molecular crowding in vivo that is dynamically regulated at the molecular level.

Cell cycle-dependent phosphorylation of MAN1.

The LEM (LAP2beta, Emerin, and MAN1) proteins are essential for nuclear membrane targeting to chromatin via an association with barrier-to-autointegration factor (BAF). Herein, we focused on the mitotic phosphorylation of MAN1 and its biological role. MAN1 was phosphorylated in a cell cycle-dependent manner in the Xenopus egg cell-free system, and the mitotic phosphorylation at the N-terminal region of MAN1 suppressed the binding of MAN1 to BAF. Titansphere column chromatography followed by MS/MS sequencing identified at least three M-phase-specific phosphorylation sites, Thr-209, Ser-351, and Ser-402, and one cell cycle-independent phosphorylation site, Ser-463. An in vitro BAF binding assay involving mutants S402A and S402E suggested that the phosphorylation of Ser-402 was important for regulation of the binding of MAN1 to BAF.

Single-molecule anatomy by atomic force microscopy and recognition imaging.

Atomic force microscopy (AFM) has been a useful technique to visualize cellular and molecular structures at single-molecule resolution. The combination of imaging and force modes has also allowed the characterization of physical properties of biological macromolecules in relation to their structures. Furthermore, recognition imaging, which is obtained under the TREC(TM) (Topography and RECognition) mode of AFM, can map a specific protein of interest within an AFM image. In this study, we first demonstrated structural properties of purified α Actinin-4 by conventional AFM. Since this molecule is an actin binding protein that cross-bridges actin filaments and anchors it to integrin via tailin-vinculin-α actinin adaptor-interaction, we investigated their structural properties using the recognition mode of AFM. For this purpose, we attached an anti-α Actinin-4 monoclonal antibody to the AFM cantilever and performed recognition imaging against α Actinin-4. We finally succeeded in mapping the epitopic region within the α Actinin-4 molecule. Thus, recognition imaging using an antibody coupled AFM cantilever will be useful for single-molecule anatomy of biological macromolecules and structures.

Nuclear matrix contains novel WD-repeat and disordered-region-rich proteins.

To find novel proteins predicted to participate in the formation of nuclear bodies, nuclear speckles, and nuclear macro-protein complexes, we applied proteome analysis to a HeLa cell nuclear matrix fraction. Proteins in the fraction were separated by SDS-PAGE, digested with trypsin, and analyzed by nanoflow liquid chromatography-iontrap-tandem mass spectrometry. Three hundred and thirty three proteins including 39 novel ones were identified. Seven WD-repeat proteins and 16 disordered region-rich proteins, which act frequently as scaffolding proteins for macro-protein complexes, were found amongst the novel proteins.