Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Membrane remodeling is required for dynamic cellular processes such as cell division, polarization and motility. BAR domain proteins and dynamins are key molecules in membrane remodeling that work together for membrane deformation and fission. In striated muscles, sarcolemmal invaginations termed T-tubules are required for excitation-contraction coupling. BIN1 and DNM2, which encode a BAR domain protein BIN1 and dynamin 2, respectively, have been reported to be causative genes of centronuclear myopathy (CNM), a hereditary degenerative disease of skeletal muscle, and deformation of T-tubules is often observed in the CNM patients. However, it remains unclear how BIN1 and dynamin 2 are implicated in T-tubule biogenesis, and how mutations in these molecules cause CNM to develop.Here, using an in cellulo reconstitution assay, we demonstrate that dynamin 2 is required for stabilization of membranous structures equivalent to T-tubules. GTPase activity of wild type dynamin 2 is suppressed through interaction with BIN1, whereas that of the disease-associated mutant dynamin 2 remains active due to lack of the BIN1-mediated regulation thus causing aberrant membrane remodeling. Finally, we show that in cellulo aberrant membrane remodeling by mutant dynamin 2 variants is correlated with their enhanced membrane fission activities, and the results can explain severity of the symptoms in patients. Thus, this study provides molecular insights into dysregulated membrane remodeling triggering the pathogenesis of DNM2-related centronuclear myopathy.

DOI： 10.1074/jbc.RA120.015184

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：eLife Sciences Publications Ltd  

Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin-mediated endocytosis. Dynamin forms helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulate membrane remodeling during the fission, but its precise mechanism remains elusive. In this study, we analyzed structural changes of dynamin-amphiphysin complexes during the membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin helices are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our approaches using combination of EM and HS-AFM clearly demonstrate new mechanistic insights into the dynamics of dynamin-amphiphysin complexes during membrane fission.

DOI： 10.7554/eLife.30246

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Cancer cell invasion is mediated by actin-based membrane protrusions termed invadopodia. Invadopodia consist of "core" F-actin bundles associated with adhesive and proteolytic machineries promoting cell invasion by degrading extracellular matrix (ECM). Formation of the F-actin core in invadopodia is regulated by various actin-binding proteins including Arp2/3 complex and cortactin. Dynamin GTPase localizes to the invadopodia and is implicated in cancer cell invasion, but its precise role at the invadopodia remained elusive.
In this study, we examined the roles of dynamin at the invadopodia of bladder cancer cells. Although all three dynamin isoforms (dynamin1, 2 and 3) are expressed in human bladder cancer cell line T24, only dynamin2 localizes to the invadopodia. Inhibition of dynamin2 function, using either RNA interference (RNAi) or the dynamin specific inhibitor Dynasore, caused defects in invadopodia formation and suppressed invasive activity of 124 bladder cancer cells. Structure-function analysis using dynamin2 deletion fragments identified the proline/arginine-rich domain (PRD) of dynamin2 as indispensable for invadopodia formation and invasiveness of 124 cells. Thus, dynamin2 contributes to bladder cancer invasion by controlling invadopodia formation in bladder cancer cells and may prove a valuable therapeutic target. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2016.10.063

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC  

Cytokinesis is a highly ordered cellular process driven by interactions between central spindle microtubules and the actomyosin contractile ring linked to the dynamic remodelling of the plasma membrane. The mechanisms responsible for reorganizing the plasma membrane at the cell equator and its coupling to the contractile ring in cytokinesis are poorly understood. We report here that Syndapin, a protein containing an F-BAR domain required for membrane curvature, contributes to the remodelling of the plasma membrane around the contractile ring for cytokinesis. Syndapin colocalizes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) at the cleavage furrow, where it directly interacts with a contractile ring component, Anillin. Accordingly, Anillin is mislocalized during cytokinesis in Syndapin mutants. Elevated or diminished expression of Syndapin leads to cytokinesis defects with abnormal cortical dynamics. The minimal segment of Syndapin, which is able to localize to the cleavage furrow and induce cytokinesis defects, is the F-BAR domain and its immediate C-terminal sequences. Phosphorylation of this region prevents this functional interaction, resulting in reduced ability of Syndapin to bind to and deform membranes. Thus, the dephosphorylated form of Syndapin mediates both remodelling of the plasma membrane and its proper coupling to the cytokinetic machinery.

DOI： 10.1098/rsob.130081

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Specialized membrane domains containing lipid rafts are thought to be important for membrane processes such as signaling and trafficking [1, 2]. An unconventional type I myosin has been shown to reside in lipid rafts and function to target a disaccharidase to rafts in brush borders of intestinal mammalian cells [3]. In the fission yeast Schizosaccharomyces pombe, distinct sterol-rich membrane domains are formed at the cell division site and sites of polarized cell growth at cell tips [4]. Here, we show that the sole S. pombe myosin I, myo1p, is required for proper organization of those membrane domains. myo1 mutants lacking the TH1 domain exhibit a uniform distribution of sterol-rich membranes all over the plasma membrane throughout the cell cycle. These effects are independent of endocytosis because myo1 mutants exhibit no endocytic defects. Conversely, overexpression of myo1p induces ectopic sterol-rich membrane domains. Myo1p localizes to nonmotile foci that cluster in sterol-rich plasma membrane domains and fractionates with detergent-resistant membranes. Because the myo1p TH1 domain may bind directly to acidic: phospholipids, these findings suggest a model for how type I myosin contributes to the organization of specialized membrane domains.

DOI： 10.1016/j.cub.2005.07.009

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Many membrane processes occur in discrete membrane domains containing lipid rafts(1), but little is known about how these domains are organized and positioned. In the fission yeast Schizosaccharomyces pombe, a sterol-rich membrane domain forms at the cell-division site(2). Here, we show that formation of this membrane domain is independent of the contractile actin ring, septation, mid1p and the septins, and also requires cdc15p(3), an essential contractile ring protein that associates with lipid rafts. cdc15 mutants have membrane domains in the shape of spirals. Overexpression of cdc15p in interphase cells induces abnormal membrane domain formation in an actin-independent manner. We propose that cdc15p functions to organize lipid rafts at the cleavage site for cytokinesis.

DOI： 10.1038/ncb1189

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Publishing type：Research paper (scientific journal)   Publisher：AIP Publishing  

High-speed atomic force microscopy (HS-AFM) is a powerful tool for studying the dynamics of biomolecules in vitro because of its high temporal and spatial resolution. However, multi-functionalization, such as combination with complementary measurement methods, environment control, and large-scale mechanical manipulation of samples, is still a complex endeavor due to the inherent design and the compact sample scanning stage. Emerging tip-scan HS-AFM overcame this design hindrance and opened a door for additional functionalities. In this study, we designed a motor-driven stretching device to manipulate elastic substrates for HS-AFM imaging of biomolecules under controllable mechanical stimulation. To demonstrate the applicability of the substrate stretching device, we observed a microtubule buckling by straining the substrate and actin filaments linked by α-actinin on a curved surface. In addition, a BAR domain protein BIN1 that senses substrate curvature was observed while dynamically controlling the surface curvature. Our results clearly prove that large-scale mechanical manipulation can be coupled with nanometer-scale imaging to observe biophysical effects otherwise obscured.

DOI： 10.1063/5.0111017

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Language：English   Publishing type：Research paper (scientific journal)  

Dynamin is an endocytic protein that functions in vesicle formation by scission of invaginated membranes. Dynamin maintains the structure of foot processes in glomerular podocytes by directly and indirectly interacting with actin filaments. However, molecular mechanisms underlying dynamin-mediated actin regulation are largely unknown. Here, biochemical and cell biological experiments were conducted to uncover how dynamin modulates interactions between membranes and actin in human podocytes. Actin-bundling, membrane tubulating, and GTPase activities of dynamin were examined in vitro using recombinant dynamin 2-wild-type (WT) or dynamin 2-K562E, which is a mutant found in Charcot-Marie-Tooth patients. Dynamin 2-WT and dynamin 2-K562E led to the formation of prominent actin bundles with constant diameters. Whereas liposomes incubated with dynamin 2-WT resulted in tubule formation, dynamin 2-K562E reduced tubulation. Actin filaments and liposomes stimulated dynamin 2-WT GTPase activity by 6- and 20-fold, respectively. Actin-filaments, but not liposomes, stimulated dynamin 2-K562E GTPase activity by 4-fold. Self-assembly-dependent GTPase activity of dynamin 2-K562E was reduced to one-third compared to that of dynamin 2-WT. Incubation of liposomes and actin with dynamin 2-WT led to the formation of thick actin bundles, which often bound to liposomes. The interaction between lipid membranes and actin bundles by dynamin 2-K562E was lower than that by dynamin 2-WT. Dynamin 2-WT partially colocalized with stress fibers and actin bundles based on double immunofluorescence of human podocytes. Dynamin 2-K562E expression resulted in decreased stress fiber density and the formation of aberrant actin clusters. Dynamin 2-K562E colocalized with α-actinin-4 in aberrant actin clusters. Reformation of stress fibers after cytochalasin D-induced actin depolymerization and washout was less effective in dynamin 2-K562E-expressing cells than that in dynamin 2-WT. Bis-T-23, a dynamin self-assembly enhancer, was unable to rescue the decreased focal adhesion numbers and reduced stress fiber density induced by dynamin 2-K562E expression. These results suggest that the low affinity of the K562E mutant for lipid membranes, and atypical self-assembling properties, lead to actin disorganization in HPCs. Moreover, lipid-binding and self-assembly of dynamin 2 along actin filaments are required for podocyte morphology and functions. Finally, dynamin 2-mediated interactions between actin and membranes are critical for actin bundle formation in HPCs.

DOI： 10.3389/fcell.2022.884509

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Language：English   Publishing type：Research paper (scientific journal)  

Dynamin 1 is a neuronal endocytic protein that participates in vesicle formation by scission of invaginated membranes. Dynamin 1 is also expressed in the kidney; however, its physiological significance to this organ remains unknown. Here, we show that dynamin 1 is crucial for microtubule organization and stabilization in glomerular podocytes. By immunofluorescence and immunoelectron microscopy, dynamin 1 was concentrated at microtubules at primary processes in rat podocytes. By immunofluorescence of differentiated mouse podocytes (MPCs), dynamin 1 was often colocalized with microtubule bundles, which radially arranged toward periphery of expanded podocyte. In dynamin 1-depleted MPCs by RNAi, α-tubulin showed a dispersed linear filament-like localization, and microtubule bundles were rarely observed. Furthermore, dynamin 1 depletion resulted in the formation of discontinuous, short acetylated α-tubulin fragments, and the decrease of microtubule-rich protrusions. Dynamins 1 and 2 double-knockout podocytes showed dispersed acetylated α-tubulin and rare protrusions. In vitro, dynamin 1 polymerized around microtubules and cross-linked them into bundles, and increased their resistance to the disassembly-inducing reagents Ca2+ and podophyllotoxin. In addition, overexpression and depletion of dynamin 1 in MPCs increased and decreased the nocodazole resistance of microtubules, respectively. These results suggest that dynamin 1 supports the microtubule bundle formation and participates in the stabilization of microtubules.

DOI： 10.1096/fj.202001240RR

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Language：Japanese   Publisher：(一社)日本細胞生物学会  

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Language：English   Publishing type：Research paper (scientific journal)  

Subsequently to the publication of the above article, the authors have realized that the second‑listed author, The Mon La, had not been properly credited as one of the co‑writers of the paper. Therefore, the Authors' Contributions of the Declarations section of the article should have read as follows: Authors' contributions HY, KTa and TML designed the research and wrote the paper. HY, TA, YM, EO and TT performed mutant protein construction, protein purification and actin bundling experiments. TA and YM performed electron microscopy. EO, TML, KS and KF performed immunofluorescent microscopy, cell migration assay and analyzed data. FYW and KTo identified phosphorylation sites by MALDI‑MS. All authors read and approved the final manuscript. The authors apologize to the readership of the Journal for the misinformation in this regard, and for any inconvenience caused. [the original article was published in International Journal of Oncology 54: 550‑558, 2019; DOI: 10.3892/ijo.2018.4663].

DOI： 10.3892/ijo.2020.4962

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Language：English   Publishing type：Research paper (scientific journal)  

Dynamin copolymerizes with cortactin to form a ring‑like complex that bundles and stabilizes actin filaments. Actin bundle formation is crucial for generation of filopodia and lamellipodia, which guide migration, invasion, and metastasis of cancer cells. However, it is unknown how the dynamin‑cortactin complex regulates actin bundle formation. The present study investigated phosphorylation of cortactin by cyclin‑dependent kinase 5 (CDK5) and its effect on actin bundle formation by the dynamin‑cortactin complex. CDK5 directly phosphorylated cortactin at T145/T219 in vitro. Phosphomimetic mutants in which one or both of these threonine residues was substituted by aspartate were used. The three phosphomimetic mutants (T145D, T219D and T145DT219D) had a decreased affinity for F‑actin. Furthermore, electron microscopy demonstrated that these phosphomimetic mutants could not form a ring‑like complex with dynamin 1. Consistently, the dynamin 1‑phosphomimetic cortactin complexes exhibited decreased actin‑bundling activity. Expression of the phosphomimetic mutants resulted in not only aberrant lamellipodia and short filopodia but also cell migration in NG108‑15 glioma‑derived cells. These results indicate that phosphorylation of cortactin by CDK5 regulates formation of lamellipodia and filopodia by modulating dynamin 1/cortactin‑dependent actin bundling. Taken together, these findings suggest that CDK5 is a potential molecular target for anticancer therapy.

DOI： 10.3892/ijo.2018.4663

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Language：Japanese   Publisher：(公社)日本生物工学会  

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Language：Japanese   Publisher：日本筋学会  

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Language：English   Publisher：生命科学系学会合同年次大会運営事務局  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

The endocytic protein dynamin participates in the formation of actin-based membrane protrusions such as podosomes, pseudopodia, and invadopodia, which facilitate cancer cell migration, invasion, and metastasis. However, the role of dynamin in the formation of actin-based membrane protrusions at the leading edge of cancer cells is unclear. In this study, we demonstrate that the ubiquitously expressed dynamin 2 isoform facilitates cell migration by stabilizing F-actin bundles in filopodia of the lung cancer cell line H1299. Pharmacological inhibition of dynamin 2 decreased cell migration and filopodial formation. Furthermore, dynamin 2 and cortactin mostly colocalized along F-actin bundles in filopodia of serum-stimulated H1299 cells by immunofluorescent and immunoelectron microscopy. Knockdown of dynamin 2 or cortactin inhibited the formation of filopodia in serum-stimulated H1299 cells, concomitant with a loss of F-actin bundles. Expression of wild-type cortactin rescued the punctate-like localization of dynamin 2 and filopodial formation. The incubation of dynamin 2 and cortactin with F-actin induced the formation of long and thick actin bundles, with these proteins colocalizing at F-actin bundles. A depolymerization assay revealed that dynamin 2 and cortactin increased the stability of F-actin bundles. These results indicate that dynamin 2 and cortactin participate in cell migration by stabilizing F-actin bundles in filopodia. Taken together, these findings suggest that dynamin might be a possible molecular target for anticancer therapy.

DOI： 10.3892/ijo.2016.3592

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Specific mutations in dynamin 2 are linked to Charcot-Marie-Tooth disease (CMT), an inherited peripheral neuropathy. However, the effects of these mutations on dynamin function, particularly in relation to the regulation of the actin cytoskeleton remain unclear. Here, selected CMT-associated dynamin mutants were expressed to examine their role in the pathogenesis of CMT in U2OS cells. Ectopic expression of the dynamin CMT mutants 555 Delta 3 and K562E caused an approximately 50% decrease in serum stimulation dependent lamellipodia formation; however, only K562E caused aberrations in the actin cytoskeleton. Immunofluorescence analysis showed that the K562E mutation resulted in the disappearance of radially aligned actin bundles and the simultaneous appearance of F-actin clusters. Live-cell imaging analyses showed F-actin polymers of decreased length assembled into immobile clusters in K562E-expressing cells. The K562E dynamin mutant colocalized with the F-actin clusters, whereas its colocalization with clathrin-coated pit marker proteins was decreased. Essentially the same results were obtained using another cell line, HeLa and NG108-15 cells. The present study is the first to show the association of dynamin CMT mutations with aberrant actin dynamics and lamellipodia, which may contribute to defective endocytosis and myelination in Schwann cells in CMT. (C) 2016 The Authors. Published by Elsevier Ireland Ltd.

DOI： 10.1016/j.neulet.2016.06.030

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Background Information. Cortactin contributes to growth cone morphogenesis by forming with dynamin, ring-shaped complexes that mechanically bundle and stabilise F-actin. However, the regulatory mechanism of cortactin action is poorly understood.
Results. Immunofluorescence microscopy revealed that protein kinase C (PKC) colocalises with cortactin at growth cone filopodia in SH-SY5Y neuroblastoma cells. PKC activation by phorbol 12-myristate 13-acetate causes cortactin phosphorylation, filopodial retraction and F-actin-bundle loss. Moreover, PKC directly phosphorylates cortactin in vitro at S135/T145/S172, mitigating both cortactin's actin-binding and actin-crosslinking activity, whereas cellular expression of a phosphorylation-mimetic cortactin mutant hinders filopodial formation with a significant decrease of actin bundles.
Conclusions. Our results indicate that PKC-mediated cortactin phosphorylation might be implicated in the maintenance of growth cone.

DOI： 10.1111/boc.201500032

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC  

Cytokinesis controls the proper segregation of nuclear and cytoplasmic materials at the end of cell division. The chromosomal passenger complex (CPC) has been proposed to monitor the final separation of the two daughter cells at the end of cytokinesis in order to prevent cell abscission in the presence of DNA at the cleavage site, but the precise molecular basis for this is unclear. Recent studies indicate that abscission could be mediated by the assembly of filaments comprising components of the endosomal sorting complex required for transport-III (ESCRT-III). Here, we show that the CPC subunit Borealin interacts directly with the Snf7 components of ESCRT-III in both Drosophila and human cells. Moreover, we find that the CPC's catalytic subunit, Aurora B kinase, phosphorylates one of the three human Snf7 paralogues-CHMP4C-in its C-terminal tail, a region known to regulate its ability to form polymers and associate with membranes. Phosphorylation at these sites appears essential for CHMP4C function because their mutation leads to cytokinesis defects. We propose that CPC controls abscission timing through inhibition of ESCRT-III Snf7 polymerization and membrane association using two concurrent mechanisms: interaction of its Borealin component with Snf7 proteins and phosphorylation of CHMP4C by Aurora B.

DOI： 10.1098/rsob.120070

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

Microtubules maintain an intimate relationship with the rings of anillin, septins and actomyosin filaments throughout cytokinesis. in Drosophila, peripheral microtubules emanating from the spindle poles contact the equatorial cell cortex to deliver the signal that initiates formation of the cytokinetic furrow. Mutations that affect microtubule stability lead to ectopic furrowing because peripheral microtubules contact inappropriate cortical sites. The PAV-KLP (Pavarotti-kinesin-like protein)/RacGAP50C (where GAP is GTPase-activating protein) centralspindlin complex moves towards the plus ends of microtubules to reach the cell equator. When RacGAP50C is tethered to the cell membrane, furrowing initiates at multiple non-equatorial sites, indicating that mis-localization of this single molecule is sufficient to promote furrowing. Furrow formation and ingression requires RhoA activation by the RhoGEF (guanine-nucleotide-exchange factor) Pebble, which interacts with RacGAP50C. RacGAP50C also binds anillin, which associates with actin, myosin and septins. Thus RacGAP50C plays a pivotal role during furrow formation by activating RhoA and linking the peripheral microtubules with the nascent rings through its interaction with anillin.

DOI： 10.1042/BST0360400

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

Formins are conserved actin nucleators which promote the assembly of actin filaments for the formation of diverse actin structures. In fission yeast Schizosaccharomyces pombe, the formin cdc12p is required specifically in assembly of the actin-based contractile ring during cytokinesis. Here, using a mutational analysis of cdc12p, we identify regions of cdc12p responsible for ring assembly and localization. Profilin-binding residues of the FH1 domain regulate actin assembly and processive barbed-end capping by the FH2 domain. Studies using photobleaching ( FRAP) and sensitivity to latrunculin A treatment show that profilin binding modulates the rapid dynamics of actin and cdc12p within the ring in vivo. Visualized by functional GFP-fusion constructs expressed from the endogenous promoter, cdc12p appears in a small number of cytoplasmic motile spot structures that deliver the formin to the ring assembly site, without detectable formation of an intermediate band of "nodes." The FH3/DID region directs interphase spot localization, while an N-terminal region and the FH1-FH2 domains of cdc12p can target its localization to the ring. Mutations in putative DID and DAD regions do not alter regulation, suggesting that cdc12p is not regulated by a canonical autoinhibition mechanism. Our findings provide insights into the regulation of formin activity and the mechanisms of contractile ring dynamics and assembly.

DOI： 10.1091/mbc.E07-07-0731

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Anillin, one of the first factors recruited to the cleavage site during cytokinesis, interacts with actin, myosin II and septins, and is essential for proper organization of the actomyosin contractile ring. We employed affinity-purification methodology coupled with mass spectrometry to identify Anillin-interacting molecules in Drosophila cells. We isolated several actin and myosin proteins, three of the five Drosophila septins and RacGAP50C (Tum), a component of the centralspindlin complex. Using drug and RNA interference (RNAi) treatments we established that F-actin is essential for Anillin cortical localization in prometaphase but not for its accumulation at the cleavage furrow after anaphase onset. Moreover, septins were not recruited to the cleavage site in cells in which Anillin was knocked down by RNAi, but localized to central-spindle microtubules, suggesting that septins travel along microtubules to interact with Anillin at the furrow. Finally, we demonstrate that RacGAP50C is necessary for Anillin accumulation at the furrow and that the two proteins colocalize in vivo and interact in vitro. Thus, in addition to its role in activating RhoA signalling, RacGAP50C also controls the proper assembly of the actomyosin ring by interacting with Anillin at the cleavage furrow.

DOI： 10.1242/jcs.026716

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Calmodulin (CaM) is known to be a ciliary component. However, the function of CaM in cilia or flagella has not been well understood. Immunoelectron microscopy using anti-CaM antibody showed that CaM was localized on the axonemal microtubules (MTs) and matrix of Tetrahymena cilia. To investigate the signal transduction of Ca2+/CaM in cilia, we performed Ca2+/CaM-affinity column chromatography in the membrane and matrix fraction. Elongation factor-lot (EF-1alpha) was identified as a Ca2+/CaM-binding protein in cilia. EF-1alpha is a highly conserved protein and functions in protein translation. In addition, EF-1alpha has been reported to interact with MTs and F-actin in several organisms. Immunoelectron microscopy showed that EF-1alpha was localized on the axonemal MTs. However, in immunoblot analysis, EF-1alpha was mainly extracted in the membrane and matrix fraction from the axonemal MTs by 1% Triton X-100 extraction. These results suggest that interaction between EF-1alpha and axonemal MTs is weak and sensitive to treatment with 1% Triton X-100 and that EF-1alpha mediates between axonemal MTs and CaM in the presence of Ca2+. Moreover, EF-1alpha was also localized in cilia of Paramecium, suggesting that EF-1alpha functions as a target protein of Ca2+/CaM in ciliate cilia. (C) 2003 Wiley-Liss, Inc.

DOI： 10.1002/cm.10111

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER THORACIC SOC  

Asthma is characterized by reversible airway obstruction and airway inflammation. Serum levels of eosinophil cationic protein (ECP) might reflect eosinophilic airway inflammation and asthma activity. However, serum ECP levels are not elevated in some patients with asthma, even when they are symptomatic. In this study, we screened for polymorphisms in the ECP gene and analyzed association between these polymorphisms and asthma and serum ECP levels in 137 Japanese families identified through children with asthma. We identified three polymorphisms (-393C/T, -38C/A, and 124Arg/Thr) in human ECP. We did not find associations between these polymorphisms and asthma by the transmission disequilibrium test. However, we found that serum ECP levels in subjects with the -393T allele were significantly lower than those in subjects with the -393C allele. A reporter construct with the -393T allele showed significantly lower promoter activity than one with the -393C allele. Gel shift assay revealed that C/EBP proteins can bind the -393C/T polymorphic site. These data indicate that C/EBP proteins play an important role in the regulation of ECP and that a significant amount of the variance in baseline serum ECP levels may be explained by the -393C/T polymorphism. Although ECP polymorphisms are not likely to be involved in the development of asthma, measurement of ECP levels for the assessment of asthma activity may be improved when done in combination with genotyping of the -393C/T polymorphism.

DOI： 10.1164/rccm.200204-292OC

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

Background: Tetrahymena 14-nm filament protein (14FP) is bifunctional, with roles as a citrate synthase in mitochondria and as a cytoskeletal protein in nuclear events during fertilization and in oral morphogenesis, In this study, to further our understanding of the bifunctional property of 14FP, we attempted to screen 14FP-binding proteins using affinity column chromatography,
Results: Through the screening of 14FP-binding proteins using 14FP-affinity chromatography, we detected 65 kDa and 70 kDa proteins that bound to 14FP in an ATP dependent manner. From the N-terminal amino acid sequence, these proteins were identified as the Tetrahymena mitochondrial chaperones, hsp60 and mthsp70, respectively. Tetrahymena hsp60 was recognized with a monoclonal antibody raised against human hsp60. Immunofluorescence and immunoelectron microscopy using the monoclonal antibody showed that Tetrahymena hsp60 was localized to mitochondria, Moreover, Tetrahymena hsp60 was also present at extramitochondrial sites including basal bodies of cilia and oral apparatus, and particularly at the developing oral apparatus during cell division.
Conclusion: These results suggest that Tetrahymena hsp60 is localized in basal bodies and is involved in cortical patterning such as the formation of the oral apparatus as well as having a role in the folding of mitochondrial proteins in mitochondria.

DOI： 10.1046/j.1365-2443.2001.00400.x

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

Tetrahymena 14-nm filament protein/citrate synthase (49K protein) is a bifunctional protein with roles in the cytoskeleton and as a citrate synthase. Though previous studies have shown that the 49K protein is derived from a single transcript of a single gene, direct demonstration of the 49K protein's bifunctional property remained to be elucidated. In this study, a recombinant 49K protein was expressed in Escherichia coli, purified and characterized. The citrate synthase activity of the recombinant 49K protein was comparable to that of the 49K protein purified from Tetrahymena. The recombinant 49K protein formed 14-nm filaments, but only of short length. The filaments were elongated in the presence of a soluble fraction of Tetrahymena. These results suggest that the 49K protein itself is bifunctional, but some co-factor(s) is necessary for elongation of filaments. (C) 1997 Academic Press.

DOI： 10.1006/bbrc.1997.7008

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN BIOCHEMICAL SOC  

Tetrahymena 14-nm filament protein (49K protein) is a bifunctional protein with roles in the cytoskeleton and as citrate synthase. Previous studies in our laboratory showed that elongation factor 1 alpha (EF-1 alpha) copurifies with the 49K protein upon polymerization and depolymerization of the 49K protein. In this study, the 49K protein was isolated from partially purified 49K protein fraction containing EF-1 alpha. Using the purified 49K protein and/or purified EF-1 alpha, the interaction between 49K protein and EF-1 alpha in filament formation was investigated electronmicroscopically and it was demonstrated that purified 49K protein was capable of forming 14-nm filaments without EF-1 alpha. The 49K protein/citrate synthase has been suggested to form filaments in mitochondria. Here we show that the citrate synthase activity of 49K protein is decreased by polymerization and increased by depolymerization, suggesting a possible modulating mechanism of citrate synthase activity by monomer-polymer conversion in mitochondria in situ.

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Authorship：Corresponding author   Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

Dynamin GTPase, an essential endocytotic protein, helically polymerizes at the neck of endocytic pits, and mechanically sever the membrane upon GTP hydrolysis. However, it is not known exactly how the dynamin disconnect the membrane. To clarify the mechanisms we analyzed structural changes of dynamin complexes during membrane fission using electron microscopy and high-speed atomic force microscopy (HS-AFM). Surprisingly, the dynamin ring complexes were clustered upon GTP hydrolysis and membrane constriction occurred at uncoated regions between the clusters, suggesting a novel mode of action of dynamin. In this commentary, we illustrate dynamin’s membrane fission models proposed thus far, and our novel “clusterase” model.

DOI： 10.2142/biophys.59.255

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Other Link： http://id.ndl.go.jp/bib/030043645

Language：Japanese   Publisher：(公社)日本生物工学会  

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

Cytokinesis is the final step in cell division essential for cell proliferation, tissue differentiation and animal reproduction. Cytokinesis is featured by its dramatic changes in cell shape by which a cell divides into two nascent cells with equal genomic background. The cell morphogenesis during cytokinesis is mainly driven by extensive reorganization of cytoskeleton. However, recent studies demonstrated that various membrane dynamics such as membrane trafficking and membrane remodeling also play essential roles during cytokinesis. In this review, I will overview function and regulation of the membrane dynamics during cytokinesis and discuss about its future perspectives.

DOI： 10.2142/biophys.56.013

CiNii Article

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2016206968

J-GLOBAL

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J-GLOBAL

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Language：English   Publisher：(公社)日本生化学会  

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J-GLOBAL

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J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CELL BIOLOGY  

Web of Science

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CELL BIOLOGY  

Web of Science

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CELL BIOLOGY  

Web of Science

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：ACADEMIC PRESS INC  

Web of Science

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CELL BIOLOGY  

Web of Science

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Event date： 2020.9.16 - 2020.9.18

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Event date： 2020.6.9 - 2020.6.11

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Event date： 2020.1.10 - 2020.1.12

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2019.12.3 - 2019.12.6

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Language：Japanese   Presentation type：Poster presentation  

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Event date： 2015.7.28

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Event date： 2014.6.11 - 2014.6.13

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Event date： 2013.12.3 - 2013.12.6

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2013.4.15

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Venue：東京大学分子細胞生物学研究所  

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Event date： 2011.9.24 - 2011.9.28

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Country：Japan

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Country：Japan

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