Shusaku Kurisu : 細胞遊走研究のパラダイムシフト~二次元から三次元へ (第4章2), YODOSHA CO., LTD., Aug. 2006.
Academic Paper (Judged Full Paper):
1.
Aya Tentaku, Shusaku Kurisu, Kurumi Sejima, Toshiki Nagao, Akira Takahashi and Shigenobu Yonemura : Proximal deposition of collagen IV by fibroblasts contributes to basement membrane formation by colon epithelial cells invitro., The FEBS Journal, Vol.289, No.23, 7466-7485, 2022.
(Summary)
The basement membrane (BM) underlying epithelial tissue is a thin layer of extracellular matrix that governs tissue integrity and function. Epithelial BMs are generally assembled using BM components secreted from two origins: epithelium and stroma. Although denovo BM formation involves self-assembly processes of large proteins, it remains unclear how stroma-derived macromolecules are transported and assembled, specifically in the BM region. In this study, we established an invitro co-culture model of BM formation in which DLD-1 human colon epithelial cells were cultured on top of collagen I gel containing human embryonic OUMS-36T-2 fibroblasts as stromal cells. A distinct feature of our system is represented by OUMS-36T-2 cells which are almost exclusively responsible for synthesis of collagen IV, a major BM component. Exploiting this advantage, we found that collagen IV incorporation was significantly impaired in culture conditions where OUMS-36T-2 cells were not allowed to directly contact DLD-1 cells. Soluble collagen IV, once diluted in the culture medium, did not accumulate in the BM region efficiently. Live imaging of fluorescently tagged collagen IV revealed that OUMS-36T-2 cells deposited collagen IV aggregates directly onto the basal surface of DLD-1 cells. Collectively, these results indicate a novel mode of collagen IV deposition in which fibroblasts proximal to epithelial cells exclusively contribute to collagen IV assembly during BM formation.
Elongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid-liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.
Jenna R. Sternberg, Andrew E. Prendergast, Lucie Brosse, Yasmine Cantaut-Belarif, Olivier Thouvenin, Adeline Orts-DelImmagine, Laura Castillo, Lydia Djenoune, Shusaku Kurisu, Jonathan R. McDearmid, Pierre-Luc Bardet, Claude Boccara, Hitoshi Okamoto, Patrick Delmas and Claire Wyart : Pkd2l1 is required for mechanoception in cerebrospinal fluid-contacting neurons and maintenance of spine curvature, Nature Communications, Vol.9, No.1, 3804, 2018.
(Summary)
Defects in cerebrospinal fluid (CSF) flow may contribute to idiopathic scoliosis. However, the mechanisms underlying detection of CSF flow in the central canal of the spinal cord are unknown. Here we demonstrate that CSF flows bidirectionally along the antero-posterior axis in the central canal of zebrafish embryos. In the cfap298 mutant, reduction of cilia motility slows transport posteriorly down the central canal and abolishes spontaneous activity of CSF-contacting neurons (CSF-cNs). Loss of the sensory Pkd2l1 channel nearly abolishes CSF-cN calcium activity and single channel opening. Recording from isolated CSF-cNs in vitro, we show that CSF-cNs are mechanosensory and require Pkd2l1 to respond to pressure. Additionally, adult pkd2l1 mutant zebrafish develop an exaggerated spine curvature, reminiscent of kyphosis in humans. These results indicate that CSF-cNs are mechanosensory cells whose Pkd2l1-driven spontaneous activity reflects CSF flow in vivo. Furthermore, Pkd2l1 in CSF-cNs contributes to maintenance of natural curvature of the spine.
Miki Fukumoto, Shusaku Kurisu, Tesshi Yamada and Tadaomi Takenawa : α-Actinin-4 enhances colorectal cancer cell invasion by suppressing focal adhesion maturation., PLoS ONE, Vol.10, No.4, e0120616, 2015.
(Summary)
α-Actinins (ACTNs) are known to crosslink actin filaments at focal adhesions in migrating cells. Among the four isoforms of mammalian ACTNs, ACTN1 and ACTN4 are ubiquitously expressed. Recently, ACTN4 was reported to enhance cancer cell motility, invasion, and metastasis. However, the mechanism by which ACTN4 drives these malignant phenotypes remains unclear. Here, we show that ACTN4, but not ACTN1, induces the formation of immature focal adhesions in DLD-1 cells, leading to the rapid turnover of focal adhesions. Interestingly, zyxin (ZYX) assembly to focal adhesions was markedly decreased in ACTN4-expressing DLD-1 cells, while the recruitment of paxillin (PAX) occurred normally. On the other hand, in ACTN1-expressing DLD-1 cells, PAX and ZYX were normally recruited to focal adhesions, suggesting that ACTN4 specifically impairs focal adhesion maturation by inhibiting the recruitment of ZYX to focal complexes. Using purified recombinant proteins, we found that ZYX binding to ACTN4 was defective under conditions where ZYX binding to ACTN1 was observed. Furthermore, Matrigel invasion of SW480 cells that express high endogenous levels of ACTN4 protein was inhibited by ectopic expression of ACTN1. Altogether, our results suggest that ZYX defective binding to ACTN4, which occupies focal adhesions instead of ACTN1, induces the formation of immature focal adhesions, resulting in the enhancement of cell motility and invasion.
Shiro Suetsugu, Shusaku Kurisu and Tadaomi Takenawa : Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins., Physiological Reviews, Vol.94, No.4, 1219-1248, 2014.
(Summary)
All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes.
Kazuya Tsujita, Akihiro Kondo, Shusaku Kurisu, Junya Hasegawa, Toshiki Itoh and Tadaomi Takenawa : Antagonistic regulation of F-BAR protein assemblies controls actin polymerization during podosome formation., Journal of Cell Science, Vol.126, No.10, 2267-2278, 2013.
(Summary)
FBP17, an F-BAR domain protein, has emerged as a crucial factor linking the plasma membrane to WASP-mediated actin polymerization. Although it is well established that FBP17 has a powerful self-polymerizing ability that promotes actin nucleation on membranes in vitro, knowledge of inhibitory factors that counteract this activity in vivo is limited. Here, we demonstrate that the assembly of FBP17 on the plasma membranes is antagonized by PSTPIP2, another F-BAR protein implicated in auto-inflammatory disorder. Knockdown of PSTPIP2 in macrophage promotes the assembly of FBP17 as well as subsequent actin nucleation at podosomes, resulting in an enhancement of matrix degradation. This phenotype is rescued by expression of PSTPIP2 in a manner dependent on its F-BAR domain. Time-lapse total internal reflection fluorescence (TIRF) microscopy observations reveal that the self-assembly of FBP17 at the podosomal membrane initiates actin polymerization, whereas the clustering of PSTPIP2 has an opposite effect. Biochemical analysis and live-cell imaging show that PSTPIP2 inhibits actin polymerization by competing with FBP17 for assembly at artificial as well as the plasma membrane. Interestingly, the assembly of FBP17 is dependent on WASP, and its dissociation by WASP inhibition strongly induces a self-organization of PSTPIP2 at podosomes. Thus, our data uncover a previously unappreciated antagonism between different F-BAR domain assemblies that determines the threshold of actin polymerization for the formation of functional podosomes and may explain how the absence of PSTPIP2 causes auto-inflammatory disorder.
(Keyword)
Membrane deformation / BAR domain / Podosome / Autoinflammatory disease / WASP
Shusaku Kurisu and Tadaomi Takenawa : WASP and WAVE family proteins: friends or foes in cancer invasion?, Cancer Science, Vol.101, No.10, 2093-2104, 2010.
(Summary)
Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE) family proteins activate cells' major actin nucleating machinery, the actin-related protein 2/3 (Arp2/3) complex, leading to the formation and remodeling of cortical actin filament networks. Cortical actin regulation is critical in many aspects of cell physiology including cell-cell adhesion and cell motility, whose dysregulation is directly associated with cancer invasion and metastasis. In line with this association, the WASP and WAVE family proteins have been reported to be involved in cancer malignancies. What is puzzling, however, is that they can act as either enhancers or suppressors of cancer malignancies depending on the type of cancer and its pathological stage. We are still far from understanding the roles of the WASP and WAVE family proteins in cancer progression. Here, we summarize the recent advances of studies of the WASP and WAVE family proteins with respect to cancer invasion and we offer a model that can account for the diverse outcomes originating from dysregulated WASP and WAVE family proteins in cancer development.
(Keyword)
cancer / Cancer invasion / WASP family / metastasis
Shusaku Kurisu and Tadaomi Takenawa : The WASP and WAVE family proteins., Genome Biology, Vol.10, No.6, 226, 2009.
(Summary)
All eukaryotic cells need to reorganize their actin cytoskeleton to change shape, divide, move, and take up nutrients for survival. The Wiskott-Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous protein (WAVE) family proteins are fundamental actin-cytoskeleton reorganizers found throughout the eukaryotes. The conserved function across species is to receive upstream signals from Rho-family small GTPases and send them to activate the Arp2/3 complex, leading to rapid actin polymerization, which is critical for cellular processes such as endocytosis and cell motility. Molecular and cell biological studies have identified a wide array of regulatory molecules that bind to the WASP and WAVE proteins and give them diversified roles in distinct cellular locations. Genetic studies using model organisms have also improved our understanding of how the WASP- and WAVE-family proteins act to shape complex tissue architectures. Current efforts are focusing on integrating these pieces of molecular information to draw a unified picture of how the actin cytoskeleton in a single cell works dynamically to build multicellular organization.
(Keyword)
WASP family / actin cytoskeleton / Arp2/3 / WAVE family
Daisuke Yamazaki, Shusaku Kurisu and Tadaomi Takenawa : Involvement of Rac and Rho signaling in cancer cell motility in 3D substrates., Oncogene, Vol.28, No.13, 1570-1583, 2009.
(Summary)
The motility of cancer cells in 3D matrices is of two types: mesenchymal motility, in which the cells are elongated and amoeboid motility, in which the cells are round. Amoeboid motility is driven by an actomyosin-based contractile force, which is regulated by the Rho/ROCK pathway. However, the molecular mechanisms underlying the motility of elongated cells remain unknown. Here, we show that the motility of elongated cells is regulated by Rac signaling through the WAVE2/Arp2/3-dependent formation of elongated pseudopodia and cell-substrate adhesion in 3D substrates. The involvement of Rac signaling in cell motility was different in cell lines that displayed an elongated morphology in 3D substrates. In U87MG glioblastoma cells, most of which exhibit mesenchymal motility, inhibition of Rac signaling blocked the invasion of these cells in 3D substrates. In HT1080 fibrosarcoma cells, which display mixed cell motility involving both elongated and rounded cells, inhibition of Rac1 signaling not only blocked mesenchymal motility but also caused a mesenchymal-amoeboid transition. Additionally, Rac1 and RhoA signaling regulated the mesenchymal and amoeboid motility in these cells, respectively, and the inhibition of both pathways dramatically decreased cell invasion. Hence, we could conclude that Rac1 and RhoA signaling simultaneously regulate cell invasion in 3D matrices.
(Keyword)
cancer / Cancer invasion / Actin cytoskeleton / Cell migration
Shiro Suetsugu, Shusaku Kurisu, Tsukasa Oikawa, Daisuke Yamazaki, Atsushi Oda and Tadaomi Takenawa : Optimization of WAVE2 complex-induced actin polymerization by membrane-bound IRSp53, PIP3, and Rac., The Journal of Cell Biology, Vol.173, No.4, 571-585, 2006.
(Summary)
WAVE2 activates the actin-related protein (Arp) 2/3 complex for Rac-induced actin polymerization during lamellipodium formation and exists as a large WAVE2 protein complex with Sra1/PIR121, Nap1, Abi1, and HSPC300. IRSp53 binds to both Rac and Cdc42 and is proposed to link Rac to WAVE2. We found that the knockdown of IRSp53 by RNA interference decreased lamellipodium formation without a decrease in the amount of WAVE2 complex. Localization of WAVE2 at the cell periphery was retained in IRSp53 knockdown cells. Moreover, activated Cdc42 but not Rac weakened the association between WAVE2 and IRSp53. When we measured Arp2/3 activation in vitro, the WAVE2 complex isolated from the membrane fraction of cells was fully active in an IRSp53-dependent manner but WAVE2 isolated from the cytosol was not. Purified WAVE2 and purified WAVE2 complex were activated by IRSp53 in a Rac-dependent manner with PIP(3)-containing liposomes. Therefore, IRSp53 optimizes the activity of the WAVE2 complex in the presence of activated Rac and PIP(3).
Daisuke Yamazaki, Shusaku Kurisu and Tadaomi Takenawa : Regulation of cancer cell motility through actin reorganization., Cancer Science, Vol.96, No.7, 379-386, 2005.
(Summary)
Cell migration is a critical step in tumor invasion and metastasis, and regulation of this process will lead to appropriate therapies for treating cancer. Cancer cells migrate in various ways, according to cell type and degree of differentiation. The different types of cell migration are regulated by different mechanisms. Reorganization of the actin cytoskeleton is the primary mechanism of cell motility and is essential for most types of cell migration. Actin reorganization is regulated by Rho family small GTPases such as Rho, Rac, and Cdc42. These small GTPases transmit extracellular chemotactic signals to downstream effectors. Of these downstream effectors, Wiskott-Aldrich syndrome protein (WASP) family proteins are key regulators of cell migration. Activated WASP family proteins induce the formation of protrusive membrane structures involved in cell migration and degradation of the extracellular matrix. Inhibition of Rho family small GTPase signaling suppresses the migration and invasion of cancer cells. Thus, control of cell migration via the actin cytoskeleton provides the possibility of regulating cancer cell invasion and metastasis.
(Keyword)
cancer / metastasis / Actin cytoskeleton / Cell motility
Shusaku Kurisu, Shiro Suetsugu, Daisuke Yamazaki, Hideki Yamaguchi and Tadaomi Takenawa : Rac-WAVE2 signaling is involved in the invasive and metastatic phenotypes of murine melanoma cells., Oncogene, Vol.24, No.8, 1309-1319, 2005.
(Summary)
WAVEs (WASP-family verprolin-homologous proteins) regulate the actin cytoskeleton through activation of Arp2/3 complex. As cell motility is regulated by actin cytoskeleton rearrangement and is required for tumor invasion and metastasis, blocking actin polymerization may be an effective strategy to prevent tumor dissemination. We show that WAVEs, especially WAVE2, are essential for invasion and metastasis of melanoma cells. Malignant B16F10 mouse melanoma cells expressed more WAVE1 and WAVE2 proteins and showed higher Rac activity than B16 parental cells, which are neither invasive nor metastatic. The effect of WAVE2 silencing by RNA interference (RNAi) on the highly invasive nature of B16F10 cells was more dramatic than that of WAVE1 RNAi. Membrane ruffling, cell motility, invasion into the extracellular matrix, and pulmonary metastasis of B16F10 cells were suppressed by WAVE2 RNAi. WAVE2 RNAi also had a profound effect on invasion induced by a constitutively active form of Rac (RacCA). In addition, ectopic expression of both RacCA and WAVE2 in B16 cells resulted in further increase in the invasiveness than that observed in B16 cells expressing only RacCA. Thus, WAVE2 acts as the primary effector downstream of Rac to achieve invasion and metastasis, suggesting that suppression of WAVE2 activity holds a promise for preventing cancer invasion and metastasis.
(Keyword)
cancer / Cancer invasion / WAVE / Arp2/3 / Melanoma
Kazuhiro Kawamura, Kazunori Takano, Shiro Suetsugu, Shusaku Kurisu, Daisuke Yamazaki, Hiroaki Miki, Tadaomi Takenawa and Takeshi Endo : N-WASP and WAVE2 acting downstream of phosphatidylinositol 3-kinase are required for myogenic cell migration induced by hepatocyte growth factor., The Journal of Biological Chemistry, Vol.279, No.52, 54862-54871, 2004.
(Summary)
During skeletal muscle regeneration caused by injury, muscle satellite cells proliferate and migrate toward the site of muscle injury. This migration is mainly induced by hepatocyte growth factor (HGF) secreted by intact myofibers and also released from injured muscle. However, the intracellular machinery for the satellite cell migration has not been elucidated. To examine the mechanisms of satellite cell migration, we utilized satellite cell-derived mouse C2C12 skeletal muscle cells. HGF induced reorganization of actin cytoskeleton to form lamellipodia in C2C12 myoblasts. HGF treatment facilitated both nondirectional migration of the myoblasts in phagokinetic track assay and directional chemotactic migration toward HGF in a three-dimensional migration chamber assay. Endogenous N-WASP and WAVE2 were concentrated in the lamellipodia at the leading edge of the migrating cells. Moreover, exogenous expression of wild-type N-WASP or WAVE2 promoted lamellipodial formation and migration. By contrast, expression of the dominant-negative mutant of N-WASP or WAVE2 and knockdown of N-WASP or WAVE2 expression by the RNA interference prevented the HGF-induced lamellipodial formation and migration. When the cells were treated with LY294002, an inhibitor of phosphatidylinositol 3-kinase, the HGF-induced lamellipodial formation and migration were abrogated. These results imply that both N-WASP and WAVE2, which are activated downstream of phosphati-dylinositol 3-kinase, are required for the migration through the lamellipodial formation of C2C12 cells induced by HGF.
Shiro Suetsugu, Daisuke Yamazaki, Shusaku Kurisu and Tadaomi Takenawa : Differential roles of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for fibroblast cell migration., Developmental Cell, Vol.5, No.4, 595-609, 2003.
(Summary)
Cell migration is driven by actin polymerization at the leading edge of lamellipodia, where WASP family verprolin-homologous proteins (WAVEs) activate Arp2/3 complex. When fibroblasts are stimulated with PDGF, formation of peripheral ruffles precedes that of dorsal ruffles in lamellipodia. Here, we show that WAVE2 deficiency impairs peripheral ruffle formation and WAVE1 deficiency impairs dorsal ruffle formation. During directed cell migration in the absence of extracellular matrix (ECM), cells migrate with peripheral ruffles at the leading edge and WAVE2, but not WAVE1, is essential. In contrast, both WAVE1 and WAVE2 are essential for invading migration into ECM, suggesting that the leading edge in ECM has characteristics of both ruffles. WAVE1 is colocalized with ECM-degrading enzyme MMP-2 in dorsal ruffles, and WAVE1-, but not WAVE2-, dependent migration requires MMP activity. Thus, WAVE2 is essential for leading edge extension for directed migration in general and WAVE1 is essential in MMP-dependent migration in ECM.
Shusaku Kurisu and Shigenobu Yonemura : Fluorescent tagging of endogenous proteins and its application to protein quantification in subcellular compartments, The Cell, Vol.55, No.5, 320-322, Apr. 2023.
In homeostatic epithelial tissues, the basement membrane appears to be a quiet, motionless structure. However, during embryonic development and tissue regeneration, the basement membrane dramatically changes its distribution and shows a variety of dynamics such as compositional transition and physicochemical alterations. Recently, it has been pointed out that the shape and function of epithelial tissues is greatly influenced by the way of formation and arrangement of the basement membrane. Here, we outline the current understanding of the roles of the basement membrane dynamics in epithelial morphogenesis, and briefly introduce our approach to visualize the movement of basement membrane components.
Aya Tentaku, Shusaku Kurisu, Akira Takahashi and Shigenobu Yonemura : 上皮基底膜 IV型コラーゲンの動態解析, 第94回日本生化学会大会, Nov. 2021.
(Keyword)
extracellular matrix / collagen / Live imaging
5.
Shusaku Kurisu and Shigenobu Yonemura : Quantitative changes of actin-binding proteins in mechanical responses of epithelial cell-cell adhesion, The 73rd Annual Meeting of the Japan Society for Cell Biology, Jul. 2021.
(Keyword)
CRISPR/Cas9 / Epithelial cell-cell junction / Actin binding protein
6.
Shusaku Kurisu and Shigenobu Yonemura : The roles of cortical actin rings in the establishment of epithelial cell polarity, The 126th Annual Meeting of The Japanese Association of Anatomists, The 98th Annual Meeting of The Physiological Society of Japan, Mar. 2021.
(Keyword)
Actin ring / Epithelial cell polarity
7.
Aya Tentaku, Shusaku Kurisu, Akira Takahashi and Shigenobu Yonemura : 間質細胞由来IV型コラーゲンががん細胞に与える影響, 第262回徳島医学会学術集会, Mar. 2021.
(Keyword)
Basement membrane / Collagen Type IV
8.
Shusaku Kurisu : 上皮細胞を支える基底膜の形成, 第262回徳島医学会学術集会, Mar. 2021.
(Keyword)
Basement membrane
9.
Shusaku Kurisu and Shigenobu Yonemura : Protein quantification at epithelial cell-cell junctions reveals a differential distribution of the actin-associated components, The 72nd Annual Meeting of the Japan Society for Cell Biology, Jun. 2020.
(Keyword)
CRISPR/Cas9 / Epithelial cell-cell junction / Actin-binding protein
10.
Shusaku Kurisu and Shigenobu Yonemura : The epithelial apico-basal boundary is defined by a cortical actomyosin ring, Joint Annual Meeting of 71st JSCB & 19 th PAAJ, Jun. 2019.
Shusaku Kurisu and Shigenobu Yonemura : 蛍光タンパク質ノックインによる細胞間接着タンパク質の定量, 第4回日本メカノバイオロジー学会学術総会, Mar. 2019.
(Keyword)
cell-cell junction / CRISPR/Cas9 / knock-in
12.
Shusaku Kurisu and Shigenobu Yonemura : Determination of protein composition at epithelial cell-cell junctions by CRISPR/Cas9-mediated fluorescent protein knockin, Joint Annual Meeting of 70th JSCB and 51st JSDB co-sponsored by APDBN, Jun. 2018.
Shusaku Kurisu and Tadaomi Takenawa : IRSp53 family of I-BAR domain proteins regulates microvillius morphogenesis by sculpting the apical plasma membrane., 第65回日本細胞生物学会大会, Jun. 2013.
Determination of protein composition of the epithelial mechanosensing complex by CRISPR/Cas9-mediated fluorescent protein knock-in (Project/Area Number: 18K06932 )
Molecular insight into the motile phenotype of the leader cells in epithelial collective migration (Project/Area Number: 25860215 )