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Gilberto M Amadofilho - One of the best experts on this subject based on the ideXlab platform.
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traffic of secondary metabolites to Cell surface in the red alga laurencia dendroidea depends on a two step transport by the cytoskeleton
PLOS ONE, 2013Co-Authors: Vanessa Moura Dos Reis, Louisi De Oliveira, Raoni Moreira Ferreira Passos, Nathan B Viana, Claudia Mermelstein, Celso Santanna, Renato Crespo Pereira, Wladimir C Paradas, Fabiano L Thompson, Gilberto M AmadofilhoAbstract:In Laurencia dendroidea, halogenated secondary metabolites are primarily located in the vacuole named the corps en cerise (CC). For chemical defence at the surface level, these metabolites are intraCellularly mobilised through vesicle transport from the CC to the Cell Periphery for posterior exocytosis of these chemicals. The Cell structures involved in this specific vesicle traffic as well as the Cellular structures related to the positioning and anchoring of the CC within the Cell are not well known. Here, we aimed to investigate the role of cytoskeletal elements in both processes. Cellular and molecular assays were conducted to i) determine the ultrastructural apparatus involved in the vesicle traffic, ii) localise cytoskeletal filaments, iii) evaluate the role of different cytoskeletal filaments in the vesicle transport, iv) identify the cytoskeletal filaments responsible for the positioning and anchoring of the CC, and v) identify the transcripts related to cytoskeletal activity and vesicle transport. Our results show that microfilaments are found within the connections linking the CC to the Cell Periphery, playing an essential role in the vesicle traffic at these connections, which means a first step of the secondary metabolites transport to the Cell surface. After that, the microtubules work in the positioning of the vesicles along the Cell Periphery towards specific regions where exocytosis takes place, which corresponds to the second step of the secondary metabolites transport to the Cell surface. In addition, microtubules are involved in anchoring and positioning the CC to the Cell Periphery. Transcriptomic analysis revealed the expression of genes coding for actin filaments, microtubules, motor proteins and cytoskeletal accessory proteins. Genes related to vesicle traffic, exocytosis and membrane recycling were also identified. Our findings show, for the first time, that actin microfilaments and microtubules play an underlying Cellular role in the chemical defence of red algae.
Elizabeth J Luna - One of the best experts on this subject based on the ideXlab platform.
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supervillin slows Cell spreading by facilitating myosin ii activation at the Cell Periphery
Journal of Cell Science, 2007Co-Authors: Norio Takizawa, Mitsuo Ikebe, Reiko Ikebe, Elizabeth J LunaAbstract:During Cell migration, myosin II modulates adhesion, Cell protrusion and actin organization at the leading edge. We show that an F-actin- and membrane-associated scaffolding protein, called supervillin (SV, p205), binds directly to the subfragment 2 domains of nonmuscle myosin IIA and myosin IIB and to the N-terminus of the long form of myosin light chain kinase (L-MLCK). SV inhibits Cell spreading via an MLCK- and myosin II-dependent mechanism. Overexpression of SV reduces the rate of Cell spreading, and RNAi-mediated knockdown of endogenous SV increases it. Endogenous and EGFP-tagged SV colocalize with, and enhance the formation of, cortical bundles of F-actin and activated myosin II during early Cell spreading. The effects of SV are reversed by inhibition of myosin heavy chain (MHC) ATPase (blebbistatin), MLCK (ML-7) or MEK (U0126), but not by inhibiting Rho-kinase with Y-27632. Flag-tagged L-MLCK co-localizes in cortical bundles with EGFP-SV, and kinase-dead L-MLCK disorganizes these bundles. The L-MLCK- and myosin-binding site in SV, SV1-171, rearranges and co-localizes with mono- and di-phosphorylated myosin light chain and with L-MLCK, but not with the short form of MLCK (S-MLCK) or with myosin phosphatase. Thus, the membrane protein SV apparently contributes to myosin II assembly during Cell spreading by modulating myosin II regulation by L-MLCK.
Mitsuo Ikebe - One of the best experts on this subject based on the ideXlab platform.
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supervillin slows Cell spreading by facilitating myosin ii activation at the Cell Periphery
Journal of Cell Science, 2007Co-Authors: Norio Takizawa, Mitsuo Ikebe, Reiko Ikebe, Elizabeth J LunaAbstract:During Cell migration, myosin II modulates adhesion, Cell protrusion and actin organization at the leading edge. We show that an F-actin- and membrane-associated scaffolding protein, called supervillin (SV, p205), binds directly to the subfragment 2 domains of nonmuscle myosin IIA and myosin IIB and to the N-terminus of the long form of myosin light chain kinase (L-MLCK). SV inhibits Cell spreading via an MLCK- and myosin II-dependent mechanism. Overexpression of SV reduces the rate of Cell spreading, and RNAi-mediated knockdown of endogenous SV increases it. Endogenous and EGFP-tagged SV colocalize with, and enhance the formation of, cortical bundles of F-actin and activated myosin II during early Cell spreading. The effects of SV are reversed by inhibition of myosin heavy chain (MHC) ATPase (blebbistatin), MLCK (ML-7) or MEK (U0126), but not by inhibiting Rho-kinase with Y-27632. Flag-tagged L-MLCK co-localizes in cortical bundles with EGFP-SV, and kinase-dead L-MLCK disorganizes these bundles. The L-MLCK- and myosin-binding site in SV, SV1-171, rearranges and co-localizes with mono- and di-phosphorylated myosin light chain and with L-MLCK, but not with the short form of MLCK (S-MLCK) or with myosin phosphatase. Thus, the membrane protein SV apparently contributes to myosin II assembly during Cell spreading by modulating myosin II regulation by L-MLCK.
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the motor activity of myosin x promotes actin fiber convergence at the Cell Periphery to initiate filopodia formation
Journal of Cell Biology, 2007Co-Authors: Hiroshi Tokuo, Katsuhide Mabuchi, Mitsuo IkebeAbstract:Filopodia are actin-rich fingerlike protrusions found at the leading edge of migrating Cells and are believed to play a role in directional sensing. Previous studies have shown that myosin-X (myoX) promotes filopodia formation and that this is mediated through its ability to deliver specific cargoes to the Cell Periphery (Tokuo, H., and M. Ikebe. 2004. Biochem Biophys. Commun. 319:214–220; Zhang, H., J.S. Berg, Z. Li, Y. Wang, P. Lang, A.D. Sousa, A. Bhaskar, R.E. Cheney, and S. Stromblad. 2004. Nat. Cell Biol. 6:523–531; Bohil, A.B., B.W. Robertson, and R.E. Cheney. 2006. Proc. Natl. Acad. Sci. USA. 103:12411–12416; Zhu, X.J., C.Z. Wang, P.G. Dai, Y. Xie, N.N. Song, Y. Liu, Q.S. Du, L. Mei, Y.Q. Ding, and W.C. Xiong. 2007. Nat. Cell Biol. 9:184–192). In this study, we show that the motor function of myoX and not the cargo function is critical for initiating filopodia formation. Using a dimer-inducing technique, we find that myoX lacking its cargo-binding tail moves laterally at the leading edge of lamellipodia and induces filopodia in living Cells. We conclude that the motor function of the two-headed form of myoX is critical for actin reorganization at the leading edge, leading to filopodia formation.
Juan S Bonifacino - One of the best experts on this subject based on the ideXlab platform.
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rusc2 and wdr47 oppositely regulate kinesin 1 dependent distribution of atg9a to the Cell Periphery
Molecular Biology of the Cell, 2021Co-Authors: Carlos M Guardia, Akansha Jain, Rafael Mattera, Alex Friefeld, Juan S BonifacinoAbstract:Autophagy-related protein 9 (ATG9) is a transmembrane protein component of the autophagy machinery that cycles between the trans-Golgi network (TGN) in the perinuclear area and other compartments in the peripheral area of the Cell. In mammalian Cells, export of the ATG9A isoform from the TGN into ATG9A-containing vesicles is mediated by the adaptor protein 4 (AP-4) complex. However, the mechanisms responsible for the subsequent distribution of these vesicles to the Cell Periphery is unclear. Herein we show that the AP-4-accessory protein RUSC2 couples ATG9A-containing vesicles to the plus-end-directed microtubule motor kinesin-1 via an interaction between a disordered region of RUSC2 and the kinesin-1 light chain (KLC). This interaction is counteracted by the microtubule-associated WD40-repeat domain 47 protein (WDR47). These findings uncover a mechanism for the peripheral distribution of ATG9A-containing vesicles, involving the function of RUSC2 as a kinesin-1 adaptor and WDR47 as a negative regulator of this function.
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Redistribution of mitochondria to the Cell Periphery alters their shape.
2019Co-Authors: Carlos M Guardia, Raffaella De Pace, Aritra Sen, Amra Saric, Michal Jarnik, David A. Kolin, Ambarish Kunwar, Juan S BonifacinoAbstract:(A) Colocalization of TOM20*-SBP-GFP with 25 nM MitoTracker Red CMXRos in HeLa Cells incubated for 30 minutes at 37°C, 5% CO2. (B) Coexpression of TOM20*-SBP-GFP with HA-KIF5B*-strep causes peripheral redistribution of mitochondria and alters mitochondrial shape. (C) Coexpression of TOM20*-SBP-GFP with strep-KIFC1*-mCh causes central redistribution of mitochondria without affecting their morphology. In A–C, nuclei were stained with DAPI. The rightmost images in A–C are 3.5× magnified views of the boxed areas. Scale bars: 10 μm. (D) Box-and-whisker plots representing the fractional distance distribution (f = 95%) of mitochondria in the conditions from panels A, B, and C (see S4 Fig and Methods section for details). Summary data available as Supporting Information (S1_Data.xlsx). (E) Electron microscopy of Cells expressing TOM20*-SBP-GFP alone (top row) or with HA-KIF5B*-strep (bottom row), where rounder mitochondria with normal internal organization is shown. Images on the right are magnifications of boxed areas on the left. Left column scale bar: 4 μm; right column scale bar: 0.6 μm. (F) Quantification of the phenotype observed in (B), using mitochondrial shape descriptors [50] (see Methods section for more details). Summary data available as Supporting Information (S1_Data.xlsx). (G,H) HeLa Cells coexpressing TOM20*-SBP-GFP with mCh-KIF5B*-strep (G) (S4 Movie) or strep-KIFC1*-mCh (H) (S5 Movie) were incubated with biotin and analyzed by live-Cell imaging. Scale bar: 10 μm. Rightmost images are 4.5× enlargements of the boxed areas. GFP, green fluorescent protein; HA, hemagglutinin; KIF, kinesin superfamily; mCh, mCherry; SBP, streptavidin-binding protein; strep, streptavidin; TOM, translocase of the outer membrane.
Renato Crespo Pereira - One of the best experts on this subject based on the ideXlab platform.
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traffic of secondary metabolites to Cell surface in the red alga laurencia dendroidea depends on a two step transport by the cytoskeleton
PLOS ONE, 2013Co-Authors: Vanessa Moura Dos Reis, Louisi De Oliveira, Raoni Moreira Ferreira Passos, Nathan B Viana, Claudia Mermelstein, Celso Santanna, Renato Crespo Pereira, Wladimir C Paradas, Fabiano L Thompson, Gilberto M AmadofilhoAbstract:In Laurencia dendroidea, halogenated secondary metabolites are primarily located in the vacuole named the corps en cerise (CC). For chemical defence at the surface level, these metabolites are intraCellularly mobilised through vesicle transport from the CC to the Cell Periphery for posterior exocytosis of these chemicals. The Cell structures involved in this specific vesicle traffic as well as the Cellular structures related to the positioning and anchoring of the CC within the Cell are not well known. Here, we aimed to investigate the role of cytoskeletal elements in both processes. Cellular and molecular assays were conducted to i) determine the ultrastructural apparatus involved in the vesicle traffic, ii) localise cytoskeletal filaments, iii) evaluate the role of different cytoskeletal filaments in the vesicle transport, iv) identify the cytoskeletal filaments responsible for the positioning and anchoring of the CC, and v) identify the transcripts related to cytoskeletal activity and vesicle transport. Our results show that microfilaments are found within the connections linking the CC to the Cell Periphery, playing an essential role in the vesicle traffic at these connections, which means a first step of the secondary metabolites transport to the Cell surface. After that, the microtubules work in the positioning of the vesicles along the Cell Periphery towards specific regions where exocytosis takes place, which corresponds to the second step of the secondary metabolites transport to the Cell surface. In addition, microtubules are involved in anchoring and positioning the CC to the Cell Periphery. Transcriptomic analysis revealed the expression of genes coding for actin filaments, microtubules, motor proteins and cytoskeletal accessory proteins. Genes related to vesicle traffic, exocytosis and membrane recycling were also identified. Our findings show, for the first time, that actin microfilaments and microtubules play an underlying Cellular role in the chemical defence of red algae.
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intra Cellular storage transport and exocytosis of halogenated compounds in marine red alga laurencia obtusa
Journal of Structural Biology, 2008Co-Authors: Leonardo T Salgado, Nathan B Viana, Renato Crespo Pereira, Leonardo R Andrade, Rachel Nunes Leal, B A P Da Gama, Marcia Attias, G Amado M FilhoAbstract:The production of secondary metabolites in seaweed have been related to a capability to partition compounds into Cellular specialized storage structures, like gland Cells and the corps en cerise (CC) or cherry bodies. The possible mechanisms that bring these compounds to the thallus surface remain poorly understood. Therefore, the aim of this work is perform a characterization of the CC and determine the intra-Cellular dynamics of halogenated compounds in Laurencia obtusa. The dynamics of CC and the mechanisms related to the intra-Cellular transport of halogenated compounds were evaluated by using optical tweezers and time-lapse video microscopy. The CC were isolated and its elemental composition was characterized using X-ray microanalysis. The Cellular distribution of halogenated compounds was also demonstrated by fluorescence microscopy. Three-dimensional reconstruction technique was used to provide a visualization of the structures that connect CC to Cell Periphery. As main findings, we confirmed that the halogenated compounds are mainly found in CC and also in vesicles distributed along the cytoplasm and within the chloroplasts. We demonstrated that CC is mechanically fixed to Cell Periphery by a stalk-like connection. A vesicle transport though membranous tubular connections was seen occurring from CC to Cell wall region. We also demonstrated a process of cortical Cell death event, resulting in degradation of CC. We suggested that the vesicle transportation along membranous tubular connections and Cell death events are related to the mechanisms of halogenated compounds exudation to the thallus surface and consequently with defensive role against herbivores and fouling.