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Li, Xiao-Yan, Xiaoming Zhou, R. Grant Rowe, Yuexian Hu, David D. Schlaepfer, Dusko Ilić, Gregory Dressler, Ann Park, Jun-Lin Guan, and Stephen J. Weiss. "Snail1 controls epithelial–mesenchymal lineage commitment in focal adhesion kinase–null embryonic cells." Journal of Cell Biology 195, no. 5 (November 21, 2011): 729–38. http://dx.doi.org/10.1083/jcb.201105103.
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Mouse embryonic cells isolated from focal adhesion kinase (FAK)–null animals at embryonic day 7.5 display multiple defects in focal adhesion remodeling, microtubule dynamics, mechanotransduction, proliferation, directional motility, and invasion. To date, the ability of FAK to modulate cell function has been ascribed largely to its control of posttranscriptional signaling cascades in this embryonic cell population. In this paper, we demonstrate that FAK unexpectedly exerts control over an epithelial–mesenchymal transition (EMT) program that commits embryonic FAK-null cells to an epithelial status highlighted by the expression of E-cadherin, desmoplakin, and cytokeratins. FAK rescue reestablished the mesenchymal characteristics of FAK-null embryonic cells to generate committed mouse embryonic fibroblasts via an extracellular signal–related kinase– and Akt-dependent signaling cascade that triggered Snail1 gene expression and Snail1 protein stabilization. These findings indentify FAK as a novel regulator of Snail1-dependent EMT in embryonic cells and suggest that multiple defects in FAK−/− cell behavior can be attributed to an inappropriate commitment of these cells to an epithelial, rather than fibroblastic, phenotype.
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Yilmaz, Özlem, Patrick A. Young, Richard J. Lamont, and George E. Kenny. "Gingival epithelial cell signalling and cytoskeletal responses to Porphyromonas gingivalis invasion." Microbiology 149, no. 9 (September 1, 2003): 2417–26. http://dx.doi.org/10.1099/mic.0.26483-0.
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Porphyromonas gingivalis, an oral pathogen, can internalize within primary gingival epithelial cells (GECs) through an invasion mechanism mediated by interactions between P. gingivalis fimbriae and integrins on the surface of the GECs. Fimbriae–integrin-based signalling events were studied by fluorescence microscopy, and the subcellular localization of integrin-associated signalling molecules paxillin and focal adhesion kinase (FAK), and the architecture of the actin and microtubule cytoskeleton were examined. GECs infected with P. gingivalis for 30 min demonstrated significant redistribution of paxillin and FAK from the cytosol to cell peripheries and assembly into focal adhesion complexes. In contrast, a fimbriae-deficient mutant of P. gingivalis did not contribute substantially to activation of paxillin or FAK. After 24 h, the majority of paxillin and FAK had returned to the cytoplasm with significant co-localization with P. gingivalis in the perinuclear region. Wild-type P. gingivalis induced nucleation of actin filaments forming microspike-like protrusions and long stable microfilaments distributed throughout the cells. Fimbriae mutants promoted a rich cortical actin meshwork accompanied by membrane ruffling dispersed along the cell membrane. Remarkable disassembly and nucleation of the actin and microtubule filamentous network was observed following 24 h infection with either wild-type or fimbriae-deficient mutants of P. gingivalis. The results show that fimbriated P. gingivalis cells induce formation of integrin-associated focal adhesions with subsequent remodelling of the actin and tubulin cytoskeleton.
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Paradžik, Tina, Iva I. Podgorski, Tanja Vojvoda Zeljko, and Mladen Paradžik. "Ancient Origins of Cytoskeletal Crosstalk: Spectraplakin-like Proteins Precede the Emergence of Cortical Microtubule Stabilization Complexes as Crosslinkers." International Journal of Molecular Sciences 23, no. 10 (May 17, 2022): 5594. http://dx.doi.org/10.3390/ijms23105594.
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Adhesion between cells and the extracellular matrix (ECM) is one of the prerequisites for multicellularity, motility, and tissue specialization. Focal adhesions (FAs) are defined as protein complexes that mediate signals from the ECM to major components of the cytoskeleton (microtubules, actin, and intermediate filaments), and their mutual communication determines a variety of cellular processes. In this study, human cytoskeletal crosstalk proteins were identified by comparing datasets with experimentally determined cytoskeletal proteins. The spectraplakin dystonin was the only protein found in all datasets. Other proteins (FAK, RAC1, septin 9, MISP, and ezrin) were detected at the intersections of FAs, microtubules, and actin cytoskeleton. Homology searches for human crosstalk proteins as queries were performed against a predefined dataset of proteomes. This analysis highlighted the importance of FA communication with the actin and microtubule cytoskeleton, as these crosstalk proteins exhibit the highest degree of evolutionary conservation. Finally, phylogenetic analyses elucidated the early evolutionary history of spectraplakins and cortical microtubule stabilization complexes (CMSCs) as model representatives of the human cytoskeletal crosstalk. While spectraplakins probably arose at the onset of opisthokont evolution, the crosstalk between FAs and microtubules is associated with the emergence of metazoans. The multiprotein complexes contributing to cytoskeletal crosstalk in animals gradually gained in complexity from the onset of metazoan evolution.
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Dubois, Fatemeh, Kyle Alpha, and Christopher E. Turner. "Paxillin regulates cell polarization and anterograde vesicle trafficking during cell migration." Molecular Biology of the Cell 28, no. 26 (December 15, 2017): 3815–31. http://dx.doi.org/10.1091/mbc.e17-08-0488.
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Cell polarization and directed migration play pivotal roles in diverse physiological and pathological processes. Herein, we identify new roles for paxillin-mediated HDAC6 inhibition in regulating key aspects of cell polarization in both two-dimensional and one-dimensional matrix environments. Paxillin, by modulating microtubule acetylation through HDAC6 regulation, was shown to control centrosome and Golgi reorientation toward the leading edge, a hallmark of cell polarization to ensure directed trafficking of promigratory factors. Paxillin was also required for pericentrosomal Golgi localization and centrosome cohesion, independent of its localization to, and role in, focal adhesion signaling. In addition, we provide evidence of an accumulation of paxillin at the centrosome that is dependent on focal adhesion kinase (FAK) and identify an important collaboration between paxillin and FAK signaling in the modulation of microtubule acetylation, as well as centrosome and Golgi organization and polarization. Finally, paxillin was also shown to be required for optimal anterograde vesicular trafficking to the plasma membrane.
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Chanez, Brice, Kevin Ostacolo, Ali Badache, and Sylvie Thuault. "EB1 Restricts Breast Cancer Cell Invadopodia Formation and Matrix Proteolysis via FAK." Cells 10, no. 2 (February 13, 2021): 388. http://dx.doi.org/10.3390/cells10020388.
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Regulation of microtubule dynamics by plus-end tracking proteins (+TIPs) plays an essential role in cancer cell migration. However, the role of +TIPs in cancer cell invasion has been poorly addressed. Invadopodia, actin-rich protrusions specialized in extracellular matrix degradation, are essential for cancer cell invasion and metastasis, the leading cause of death in breast cancer. We, therefore, investigated the role of the End Binding protein, EB1, a major hub of the +TIP network, in invadopodia functions. EB1 silencing increased matrix degradation by breast cancer cells. This was recapitulated by depletion of two additional +TIPs and EB1 partners, APC and ACF7, but not by the knockdown of other +TIPs, such as CLASP1/2 or CLIP170. The knockdown of Focal Adhesion Kinase (FAK) was previously proposed to similarly promote invadopodia formation as a consequence of a switch of the Src kinase from focal adhesions to invadopodia. Interestingly, EB1-, APC-, or ACF7-depleted cells had decreased expression/activation of FAK. Remarkably, overexpression of wild type FAK, but not of FAK mutated to prevent Src recruitment, prevented the increased degradative activity induced by EB1 depletion. Overall, we propose that EB1 restricts invadopodia formation through the control of FAK and, consequently, the spatial regulation of Src activity.
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Fortin, Jessica S., Alexandre Patenaude, Rena G. Deschesnes, Marie-France Côté, Eric Petitclerc, and René C.-Gaudreault. "ASK1-P38 Pathway is Important for Anoikis Induced by Microtubule-Targeting Aryl Chloroethylureas." Journal of Pharmacy & Pharmaceutical Sciences 13, no. 2 (June 7, 2010): 175. http://dx.doi.org/10.18433/j31g6c.
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PURPOSE. We investigated the involvement of MAPK signaling in the cell death mechanisms of classical microtubule interfering agents (MIA) and aryl-3-(2-chloroethyl)ureas (CEU) acting as antimitotics, along with CEU that don’t affect directly microtubules (non-MIA CEU). METHODS.
To ascertain the activated signaling pathway profile of MIA and non-MIA CEU, Western blot, immunoprecipitation and transfection experiments were performed. RESULTS. Non-MIA CEU do not activate p38, as opposed to MIA, and the extent of ERK and JNK activation is lower than in response to MIA. The effect of MIA and non-MIA CEU on focal adhesion associated protein was also studied; MIA were shown to induce focal adhesion dismantlement associated with a sustained increase in paxillin phosphorylation and FAK cleavage, as opposed to non-MIA CEU. In addition, bcl-2 phosphorylation and AKT cleavage, induced by all MIA tested, was not observed in response to non-MIA CEU further emphasizing the differential cell death mechanisms induced by MIA and non-MIA CEU. Pharmacologic and genetic approaches emphasize that the ASK1-p38 pathway activation contributes to the cytotoxic mechanism of MIA, in contrast to non-MIA CEU. ASK1-p38 is important for increased paxillin phosphorylation and FAK cleavage, suggesting that ASK-1-p38 is an upstream event of FA structure dismantlement induced by MIA. Moreover, the endogen inhibitor of ASK-1, thioredoxin, is released from ASK-1 in response to MIA as opposed to non-MIA CEU. CONCLUSION. Our study supports that ASK1-p38 activation is an important signaling event, induced by MIA, which impairs focal adhesion structure and induces anchorage-dependent apoptosis or anoikis.
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Rodrı́guez-Fernández, José Luis, Manuel Gómez, Alfonso Luque, Nancy Hogg, Francisco Sánchez-Madrid, and Carlos Cabañas. "The Interaction of Activated Integrin Lymphocyte Function-associated Antigen 1 with Ligand Intercellular Adhesion Molecule 1 Induces Activation and Redistribution of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 in T Lymphocytes." Molecular Biology of the Cell 10, no. 6 (June 1999): 1891–907. http://dx.doi.org/10.1091/mbc.10.6.1891.
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Integrin receptors play a central role in the biology of lymphocytes, mediating crucial functional aspects of these cells, including adhesion, activation, polarization, migration, and signaling. Here we report that induction of activation of the β2-integrin lymphocyte function-associated antigen 1 (LFA-1) in T lymphocytes with divalent cations, phorbol esters, or stimulatory antibodies is followed by a dramatic polarization, resulting in a characteristic elongated morphology of the cells and the arrest of migrating lymphoblasts. This cellular polarization was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, the interaction of the activated integrin LFA-1 with its ligand intercellular adhesion molecule 1 induced the activation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK-2). FAK activation reached a maximum after 45 min of stimulation; in contrast, PYK-2 activation peaked at 30 min, declining after 60 min. Upon polarization of lymphoblasts, FAK and PYK-2 redistributed from a diffuse localization in the cytoplasm to a region close to the microtubule-organizing center in these cells. FAK and PYK-2 activation was blocked when lymphoblasts were pretreated with actin and tubulin cytoskeleton-interfering agents, indicating its cytoskeletal dependence. Our results demonstrate that interaction of the β2-integrin LFA-1 with its ligand intercellular adhesion molecule 1 induces remodeling of T lymphocyte morphology and activation and redistribution of the cytoplasmic tyrosine kinases FAK and PYK-2.
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Kuo, Hsing-Chun, Yur-Ren Kuo, Kam-Fai Lee, Meng-Chiao Hsieh, Cheng-Yi Huang, Yung-Yu Hsieh, Ko-Chao Lee, et al. "A Comparative Proteomic Analysis of Erinacine A’s Inhibition of Gastric Cancer Cell Viability and Invasiveness." Cellular Physiology and Biochemistry 43, no. 1 (2017): 195–208. http://dx.doi.org/10.1159/000480338.
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Background / Aims: Erinacine A, isolated from the ethanol extract of the Hericium erinaceus mycelium, has been demonstrated as a new alternative anticancer medicine. Drawing upon current research, this study presents an investigation of the molecular mechanism of erinacine A inhibition associated with gastric cancer cell growth. Methods: Cell viability was determined by Annexin V–FITC/propidium iodide staining and migration using a Boyden chamber assay to determine the effects of erinacine A treatment on the proliferation capacity and invasiveness of gastric cancer cells. A proteomic assay provided information that was used to identify the differentially-expressed proteins following erinacine A treatment, as well as the mechanism of its targets in the apoptotic induction of erinacine A. Results: Our results demonstrate that erinacine A treatment of TSGH 9201 cells increased cytotoxicity and the generation of reactive oxygen species (ROS), as well as decreased the invasiveness. Treatment of TSGH 9201 cells with erinacine A resulted in the activation of caspases and the expression of TRAIL. Erinacine A induction of apoptosis was accompanied by sustained phosphorylation of FAK/AKT/p70S6K and the PAK1 pathways, as well as the generation of ROS. Furthermore, the induction of apoptosis and anti-invasion properties by erinacine A could involve the differential expression of the 14-3-3 sigma protein (1433S) and microtubule-associated tumor suppressor candidate 2 (MTUS2), with the activation of the FAK/AKT/p70S6K and PAK1 signaling pathways. Conclusions: These results lead us to speculate that erinacine A may generate an apoptotic cascade in TSGH 9201 cells by activating the FAK/AKT/p70S6K/PAK1 pathway and upregulating proteins 1433S and MTUS2, providing a new mechanism underlying the anti-cancer effects of erinacine A in human gastric cancer cells.
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Xie, Zhigang, Kamon Sanada, Benjamin Adam Samuels, Heather Shih, and Li-Huei Tsai. "Serine 732 Phosphorylation of FAK by Cdk5 Is Important for Microtubule Organization, Nuclear Movement, and Neuronal Migration." Cell 114, no. 4 (August 2003): 469–82. http://dx.doi.org/10.1016/s0092-8674(03)00605-6.
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Pham, Dinh-Chuong, Yu-Chuan Chang, Shian-Ren Lin, Yuh-Ming Fuh, May-Jywan Tsai, and Ching-Feng Weng. "FAK and S6K1 Inhibitor, Neferine, Dually Induces Autophagy and Apoptosis in Human Neuroblastoma Cells." Molecules 23, no. 12 (November 28, 2018): 3110. http://dx.doi.org/10.3390/molecules23123110.
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Human neuroblastoma cancer is the most typical extracranial solid tumor. Yet, new remedial treatment therapies are demanded to overcome its sluggish survival rate. Neferine, isolated from the lotus embryos, inhibits the proliferation of various cancer cells. This study aimed to evaluate the anti-cancer activity of neferine in IMR32 human neuroblastoma cells and to expose the concealable molecular mechanisms. IMR32 cells were treated with different concentrations of neferine, followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess cell viability. In an effort to determine the molecular mechanisms in neferine-incubated IMR32 cells, cell cycle arrest, cell migration, and focal adhesion kinase (FAK), the 70-kDa ribosomal S6 kinase 1 (S6K1), poly (ADP-ribose) polymerase (PARP), caspase-3, Beclin-1, and microtubule-associated protein 1A/1B-light chain 3 (LC3) protein expressions were investigated. Neferine strongly disrupted the neuroblastoma cell growth via induction of G2/M phase arrest. Furthermore, neferine provoked autophagy and apoptosis in IMR32 cells, confirmed by p-FAK, and p-S6K1 reduction, LC3-II accumulation, Beclin-1 overexpression, and cleaved caspase-3/PARP improvement. Finally, neferine markedly retarded cell migration of neuroblastoma cancer cells. As a result, our findings for the first time showed an explicit anti-cancer effect of neferine in IMR32 cells, suggesting that neferine might be a potential candidate against human neuroblastoma cells to improve clinical outcomes with further in vivo investigation.
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Fincham, Valerie J., Valerie G. Brunton, and Margaret C. Frame. "The SH3 Domain Directs Acto-Myosin-Dependent Targeting of v-Src to Focal Adhesions via Phosphatidylinositol 3-Kinase." Molecular and Cellular Biology 20, no. 17 (September 1, 2000): 6518–36. http://dx.doi.org/10.1128/mcb.20.17.6518-6536.2000.
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ABSTRACT The v-Src oncoprotein is translocated to integrin-linked focal adhesions, where its tyrosine kinase activity induces adhesion disruption and cell transformation. We previously demonstrated that the intracellular targeting of Src is dependent on the actin cytoskeleton, under the control of the Rho family of small G proteins. However, the assembly of v-Src into focal adhesions does not require its catalytic activity or myristylation-dependent membrane association. Here, we report that the SH3 domain is essential for the assembly of focal adhesions containing the oncoprotein by mediating a switch from a microtubule-dependent, perinuclear localization to actin-associated focal adhesions; furthermore, v-Src translocation to focal adhesions requires myosin activity, at least under normal conditions when the actin cytoskeleton is being dynamically regulated. Although the SH3 domain of v-Src is also necessary for its association with focal adhesion kinase (FAK), which is often considered a likely candidate mediator of focal adhesion targeting via its carboxy-terminal targeting sequence, we show here that binding to FAK is not essential for the targeting of v-Src to focal adhesions. The p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase also associates with v-Src in an SH3-dependent manner, but in this case inhibition of PI 3-kinase activity suppressed assembly of focal adhesions containing the oncoprotein. Thus, the Src SH3 domain, which binds PI 3-kinase and which is necessary for activation of Akt downstream, is required for the actin-dependent targeting of v-Src to focal adhesions.
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Královec, Karel, Lucie Melounková, Marcela Slováková, Nikola Mannová, Miloš Sedlák, Jan Bartáček, and Radim Havelek. "Disruption of Cell Adhesion and Cytoskeletal Networks by Thiol-Functionalized Silica-Coated Iron Oxide Nanoparticles." International Journal of Molecular Sciences 21, no. 24 (December 8, 2020): 9350. http://dx.doi.org/10.3390/ijms21249350.
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One of the major obstacles that limits the use of magnetic nanoparticles in biomedical applications is their potential toxicity. In the present study, we evaluated the cytotoxic effects of thiol-functionalized silica-coated iron oxide (Fe3O4@SiO2-SH) nanoparticles using human lung epithelial cells A549. We investigated the effect of Fe3O4@SiO2-SH nanoparticles on the cell viability, proliferation, cell cycle distribution, adhesion, apoptosis, and the orientation of the cytoskeletal networks, as well as on expression of proteins involved in cell death, cell survival, and cell adhesion. We demonstrated that exposure of A549 cells to Fe3O4@SiO2-SH nanoparticles resulted in severe disruption of the actin microfilaments and microtubule cytoskeleton and reduced the size of focal adhesions. Furthermore, cell adhesion was significantly affected as well as the phosphorylation of focal adhesion kinase (FAK), extracellular-signal-regulated kinase (ERK), and p38. Our findings highlight the need for in-depth cytotoxic evaluation of nanoparticles supporting their safer use, especially in biomedical applications.
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Raghu, Hari, Neelam Sharma-Walia, Mohanan Valiya Veettil, Sathish Sadagopan, Adriana Caballero, Ramu Sivakumar, Laszlo Varga, Virginie Bottero, and Bala Chandran. "Lipid Rafts of Primary Endothelial Cells Are Essential for Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8-Induced Phosphatidylinositol 3-Kinase and RhoA-GTPases Critical for Microtubule Dynamics and Nuclear Delivery of Viral DNA but Dispensable for Binding and Entry." Journal of Virology 81, no. 15 (May 16, 2007): 7941–59. http://dx.doi.org/10.1128/jvi.02848-06.
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ABSTRACT Early during de novo infection of human microvascular dermal endothelial (HMVEC-d) cells, Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8 [HHV-8]) induces the host cell's preexisting FAK, Src, phosphatidylinositol 3-kinase (PI3-K), Rho-GTPases, Diaphanous-2 (Dia-2), Ezrin, protein kinase C-ζ, extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB signal pathways that are critical for virus entry, nuclear delivery of viral DNA, and initiation of viral gene expression. Since several of these signal molecules are known to be associated with lipid raft (LR) domains, we investigated the role of LR during KSHV infection of HMVEC-d cells. Pretreatment of cells with LR-disrupting agents methyl β-cyclo dextrin (MβCD) or nystatin significantly inhibited the expression of viral latent (ORF73) and lytic (ORF50) genes. LR disruption did not affect KSHV binding but increased viral DNA internalization. In contrast, association of internalized viral capsids with microtubules (MTs) and the quantity of infected nucleus-associated viral DNA were significantly reduced. Disorganized and disrupted MTs and thick rounded plasma membranes were observed in MβCD-treated cells. LR disruption did not affect KSHV-induced FAK and ERK1/2 phosphorylation; in contrast, it increased the phosphorylation of Src, significantly reduced the KSHV-induced PI3-K and RhoA-GTPase and NF-κB activation, and reduced the colocalizations of PI3-K and RhoA-GTPase with LRs. Biochemical characterization demonstrated the association of activated PI3-K with LR fractions which was inhibited by MβCD treatment. RhoA-GTPase activation was inhibited by PI3-K inhibitors, demonstrating that PI3-K is upstream to RhoA-GTPase. In addition, colocalization of Dia-2, a RhoA-GTPase activated molecule involved in MT activation, with LR was reduced. KSHV-RhoA-GTPase mediated acetylation and aggregation of MTs were also reduced. Taken together, these studies suggest that LRs of endothelial cells play critical roles in KSHV infection and gene expression, probably due to their roles in modulating KSHV-induced PI3-K, RhoA-GTPase, and Dia-2 molecules essential for postbinding and entry stages of infection such as modulation of microtubular dynamics, movement of virus in the cytoplasm, and nuclear delivery of viral DNA.
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Nakagawa, Ichiro, Hiroaki Inaba, Taihei Yamamura, Takahiro Kato, Shinji Kawai, Takashi Ooshima, and Atsuo Amano. "Invasion of Epithelial Cells and Proteolysis of Cellular Focal Adhesion Components by Distinct Types of Porphyromonas gingivalis Fimbriae." Infection and Immunity 74, no. 7 (July 2006): 3773–82. http://dx.doi.org/10.1128/iai.01902-05.
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ABSTRACT Porphyromonas gingivalis fimbriae are classified into six types (types I to V and Ib) based on the fimA genes encoding FimA (a subunit of fimbriae), and they play a critical role in bacterial interactions with host tissues. In this study, we compared the efficiencies of P. gingivalis strains with distinct types of fimbriae for invasion of epithelial cells and for degradation of cellular focal adhesion components, paxillin, and focal adhesion kinase (FAK). Six representative strains with the different types of fimbriae were tested, and P. gingivalis with type II fimbriae (type II P. gingivalis) adhered to and invaded epithelial cells at significantly greater levels than the other strains. There were negligible differences in gingipain activities among the six strains; however, type II P. gingivalis apparently degraded intracellular paxillin in association with a loss of phosphorylation 30 min after infection. Degradation was blocked with cytochalasin D or in mutants with fimA disrupted. Paxillin was degraded by the mutant with Lys-gingipain disrupted, and this degradation was prevented by inhibition of Arg-gingipain activity by Nα-p-tosyl-l-lysine chloromethyl ketone. FAK was also degraded by type II P. gingivalis. Cellular focal adhesions with green fluorescent protein-paxillin macroaggregates were clearly destroyed, and this was associated with cellular morphological changes and microtubule disassembly. In an in vitro wound closure assay, type II P. gingivalis significantly inhibited cellular migration and proliferation compared to the cellular migration and proliferation observed with the other types. These results suggest that type II P. gingivalis efficiently invades epithelial cells and degrades focal adhesion components with Arg-gingipain, which results in cellular impairment during wound healing and periodontal tissue regeneration.
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Rea, K., M. Sensi, A. Anichini, S. Canevari, and A. Tomassetti. "EGFR/MEK/ERK/CDK5-dependent integrin-independent FAK phosphorylated on serine 732 contributes to microtubule depolymerization and mitosis in tumor cells." Cell Death & Disease 4, no. 10 (October 2013): e815-e815. http://dx.doi.org/10.1038/cddis.2013.353.
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Wagner, Simona, Chris J. Storbeck, Kristin Roovers, Ziad Y. Chaar, Piotr Kolodziej, Marlene McKay, and Luc A. Sabourin. "FAK/src-Family Dependent Activation of the Ste20-Like Kinase SLK Is Required for Microtubule-Dependent Focal Adhesion Turnover and Cell Migration." PLoS ONE 3, no. 4 (April 2, 2008): e1868. http://dx.doi.org/10.1371/journal.pone.0001868.
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Finst, Rip J., Peter J. Kim, and Lynne M. Quarmby. "Genetics of the Deflagellation Pathway in Chlamydomonas." Genetics 149, no. 2 (June 1, 1998): 927–36. http://dx.doi.org/10.1093/genetics/149.2.927.
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Abstract Signal-induced deflagellation in Chlamydomonas involves Ca2+-activated breakage of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. In this study, we isolated 13 new deflagellation mutants that can be divided into two phenotypic classes, the Adf class and the Fa class. Cells with the Adf deflagellation phenotype are defective in acid-stimulated Ca2+ influx, but can be induced to deflagellate by treatment with nonionic detergent and Ca2+. Genetic analyses show that the five new Adf mutations, as well as the previously identified adf1 mutation, are alleles of the ADF1 gene. Mutants in the second phenotypic class, the Fa mutants, fail to deflagellate in response to any known chemical stimulus and are defective in Ca2+-activated microtubule severing. Genetic analysis of these eight new Fa strains demonstrated that they define two complementation groups, and one of these contains the previously identified fa1 mutation. Diploid analysis showed that five alleles map to the FA1 gene, whereas four alleles define a novel gene that we have named FA2. The isolation of multiple mutant alleles of each gene, generated by either ultraviolet irradiation or insertional mutagenesis, indicates that ADF1, FA1, and FA2 may be the only genes that can be identified in a loss-of-function screen. These alleles should provide a better understanding of the regulation of microtubule severing by Ca2+.
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Fan, Zhongjun, Qi Xu, Changhui Wang, Xiukun Lin, Quanbin Zhang, and Ning Wu. "A tropomyosin-like Meretrix meretrix Linnaeus polypeptide inhibits the proliferation and metastasis of glioma cells via microtubule polymerization and FAK/Akt/MMPs signaling." International Journal of Biological Macromolecules 145 (February 2020): 154–64. http://dx.doi.org/10.1016/j.ijbiomac.2019.12.158.
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Tang, Elizabeth I., Will M. Lee, and C. Yan Cheng. "Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis." Endocrinology 2016, no. 1 (January 1, 2016): 47–62. http://dx.doi.org/10.1210/en.2015-1962.
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Abstract Germ cell transport across the seminiferous epithelium during spermatogenesis requires the intricate coordination of cell junctions, signaling proteins, and both actin- and microtubule (MT)-based cytoskeletons. Although the involvement of cytoskeletons in germ cell transport has been suggested, the precise mechanism(s) remains elusive. Based on growing evidencethat actin and MT interactions underlie fundamental cellular processes, such as cell motility, it is unlikely that actin- and MT-based cytoskeletons work independently to regulate germ cell transport in the testis. Using rats treated with adjudin, a potential male contraceptive that disrupts spermatid adhesion and transport in the testis, as a study model, we show herein that actin- and MT-based cytoskeletons are both necessary for transport of spermatids and residual bodies/phagosomes across the seminiferous epithelium in adult rat testes. Analysis of intratubular expression of F-actin and tubulin revealed disruption of both actin and MT networks, concomitant with misdirected spermatids and phagosomes in rats treated with adjudin. Actin regulatory proteins, epidermal growth factor receptor pathway substrate 8 and actin-related protein 3, were mislocalized and down-regulated at the actin-rich anchoring junction between germ and Sertoli cells (apical ectoplasmicspecialization) after adjudin treatment. Nonreceptor tyrosine kinase p-FAK-Tyr407, known to regulate F-actin nucleation via actin-related protein 3, was also mislocalized and down-regulated at the apical ectoplasmic specialization, corroborating the observation of actin cytoskeleton disruption. Additionally, spatiotemporal expression of MT regulatory protein end-binding protein 1, shown to be involved in MT-actin cross talk herein, was also disrupted after adjudin treatment. In summary, spermatid/phagosome transport across the epithelium during spermatogenesis requires the coordination between actin- and MT-based cytoskeletons. (Endocrinology 157: 1644–1659, 2016)
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Akram, Rabia, Haseeb Anwar, Muhammad Shahid Javed, Azhar Rasul, Ali Imran, Shoaib Ahmad Malik, Chand Raza, et al. "Axonal Regeneration: Underlying Molecular Mechanisms and Potential Therapeutic Targets." Biomedicines 10, no. 12 (December 8, 2022): 3186. http://dx.doi.org/10.3390/biomedicines10123186.
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Axons in the peripheral nervous system have the ability to repair themselves after damage, whereas axons in the central nervous system are unable to do so. A common and important characteristic of damage to the spinal cord, brain, and peripheral nerves is the disruption of axonal regrowth. Interestingly, intrinsic growth factors play a significant role in the axonal regeneration of injured nerves. Various factors such as proteomic profile, microtubule stability, ribosomal location, and signalling pathways mark a line between the central and peripheral axons’ capacity for self-renewal. Unfortunately, glial scar development, myelin-associated inhibitor molecules, lack of neurotrophic factors, and inflammatory reactions are among the factors that restrict axonal regeneration. Molecular pathways such as cAMP, MAPK, JAK/STAT, ATF3/CREB, BMP/SMAD, AKT/mTORC1/p70S6K, PI3K/AKT, GSK-3β/CLASP, BDNF/Trk, Ras/ERK, integrin/FAK, RhoA/ROCK/LIMK, and POSTN/integrin are activated after nerve injury and are considered significant players in axonal regeneration. In addition to the aforementioned pathways, growth factors, microRNAs, and astrocytes are also commendable participants in regeneration. In this review, we discuss the detailed mechanism of each pathway along with key players that can be potentially valuable targets to help achieve quick axonal healing. We also identify the prospective targets that could help close knowledge gaps in the molecular pathways underlying regeneration and shed light on the creation of more powerful strategies to encourage axonal regeneration after nervous system injury.
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Ghosh, Mallika, Robin Lo, Charan Devarakonda, Brian Aguilera, Ivan Ivic, and Linda H. Shapiro. "Regulation of beta1 Integrin Recycling, Cell Migration and Focal Adhesion Turnover by Cell Adhesion molecule- CD13." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 102.20. http://dx.doi.org/10.4049/jimmunol.200.supp.102.20.
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Abstract CD13 is a multifunctional cell adhesion molecule that is expressed on a variety of cells where we have shown that it modulates receptor mediated endocytosis and ligand internalization to control downstream signaling pathways. In fibroblasts and epithelial cells, CD13 is localized with β1- integrin at focal adhesions (FAs), the sites of communication between the extracellular matrix and the actin cytoskeleton, prompting our exploration of potential contributions of CD13 in focal adhesion turnover, integrin endocytosis and trafficking. Phenotypically, FAs in CD13KO fibroblasts are elongated and irregular with displaced FA accessory proteins, markedly reduced actin stress fibers and fewer microtubule extensions, consistent with a link between CD13 and the control of FA dynamics. In wild type cells, CD13 and β1-integrin co internalize, traffic to Rab5+ early endosomes and recycle to the plasma membrane via Rab11a+ recycling endosomes. Pulse-chase assays with wild type and CD13 phospho tyrosine mutant confirmed that tyrosine phosphorylated CD13 must associate with the active ARF6 for β1-integrin recycling to the membrane to occur. Conversely, the absence of CD13 results in trafficking of internalized β1-integrin from early endosomes to Rab7+ late endosomes/lysosomes and a failure to return to the cell surface. Functionally, in migrating cells CD13 accumulates at the leading edge and co-localizes with IQGAP1 to regulate cell migration and adhesion. In conclusion, CD13 interacts with FAK-Src to control proper localization of focal adhesion proteins regulating focal adhesion turnover and associates with active ARF6-IQGAP1 to promote b1 integrin recycling and cell motility.
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Finst, R. J., P. J. Kim, E. R. Griffis, and L. M. Quarmby. "Fa1p is a 171 kDa protein essential for axonemal microtubule severing in Chlamydomonas." Journal of Cell Science 113, no. 11 (June 1, 2000): 1963–71. http://dx.doi.org/10.1242/jcs.113.11.1963.
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A key event in deflagellation or deciliation is the severing of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. Previous genetic analysis revealed three genes that are essential for deflagellation in Chlamydomonas. We have now identified the first of these products, Fa1p, a protein required for Ca(2+)-dependent, axonemal microtubule severing. Genetic mapping and the availability of a tagged allele allowed us to physically map the gene to the centromere-proximal domain of the mating-type locus. We identified clones of Chlamydomonas genomic DNA that rescued the Ca(2+)-dependent axonemal microtubule severing defect of fa1 mutants. The FA1 cDNA, obtained by RT-PCR, encodes a novel protein of 171 kDa, which is predicted to contain an amino-terminal coiled-coil domain and three Ca(2+)/calmodulin binding domains. By western analysis and subcellular fractionation, the FA1 product is enriched in flagellar-basal body complexes. Based on these observations and previous studies, we hypothesize that a Ca(2+)-activated, Ca(2+)-binding protein binds Fa1p leading ultimately to the activation of axonemal microtubule severing.
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Schober, Markus, Srikala Raghavan, Maria Nikolova, Lisa Polak, H. Amalia Pasolli, Hilary E. Beggs, Louis F. Reichardt, and Elaine Fuchs. "Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics." Journal of Cell Biology 176, no. 5 (February 26, 2007): 667–80. http://dx.doi.org/10.1083/jcb.200608010.
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In response to αβ1 integrin signaling, transducers such as focal adhesion kinase (FAK) become activated, relaying to specific machineries and triggering distinct cellular responses. By conditionally ablating Fak in skin epidermis and culturing Fak-null keratinocytes, we show that FAK is dispensable for epidermal adhesion and basement membrane assembly, both of which require αβ1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of αβ1 integrin in regulating cytoskeletal dynamics and orchestrating polarized keratinocyte migration out of epidermal explants. Fak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with robust, peripheral focal adhesions and microtubules. We find that without FAK, Src, p190RhoGAP, and PKL–PIX–PAK, localization and/or activation at focal adhesions are impaired, leading to elevated Rho activity, phosphorylation of myosin light chain kinase, and enhanced tensile stress fibers. We show that, together, these FAK-dependent activities are critical to control the turnover of focal adhesions, which is perturbed in the absence of FAK.
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Sisson, John C., Christine Field, Richard Ventura, Anne Royou, and William Sullivan. "Lava Lamp, a Novel Peripheral Golgi Protein, Is Required for Drosophila melanogaster Cellularization." Journal of Cell Biology 151, no. 4 (November 13, 2000): 905–18. http://dx.doi.org/10.1083/jcb.151.4.905.
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Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti–Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion.
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Beardsley, Amanda, David M. Robertson, and Liza O’Donnell. "A complex containing α6β1-integrin and phosphorylated focal adhesion kinase between Sertoli cells and elongated spermatids during spermatid release from the seminiferous epithelium." Journal of Endocrinology 190, no. 3 (September 2006): 759–70. http://dx.doi.org/10.1677/joe.1.06867.
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Spermiation is the final step of spermatogenesis and culminates in the disengagement (release) of elongated spermatids from Sertoli cells into the seminiferous tubule lumen. Spermiation failure, wherein spermatids are retained by Sertoli cells instead of releasing, occurs after hormone suppression. The mechanisms involved in spermatid disengagement and retention are not well understood. We previously showed that β1-integrin is associated with spermatids until the point of disengagement, but the ectoplasmic specialisation junction (ES) is not. The aims of this paper are to further characterise the complex that is present immediately prior to spermatid disengagement by identifying the α-integrin form dimerised with β1-integrin, localising focal adhesion kinase (FAK) and determining if microtubules are involved. Adult Sprague–Dawley rats received testosterone and oestradiol implants and an FSH antibody for 7 days to suppress testicular testosterone and FSH and induce spermiation failure. Control rats were treated with saline. Immunohistochemical analysis showed that α6-integrin and a phosphorylated form of FAK (FAK-Tyr397) are present between late spermatids and Sertoli cells after ES removal, until the point of disengagement, and both proteins remain associated with retained spermatids after spermiation failure induced by hormone suppression. Using dual-label immunofluorescence, tubulins (and thus microtubules) were observed to co-localise with ES, but were neither associated with elongated spermatids just prior to release nor with retained spermatids following hormone suppression. These results suggest that microtubules are not involved in the final release of spermatids from Sertoli cells. We conclude that spermatid release during spermiation is mediated by a ‘disengagement complex’ containing α6β1-integrin and phospho-FAK, the function of which can be affected by gonadotrophin suppression.
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Mahjoub, Moe R., Ben Montpetit, Lifan Zhao, Rip J. Finst, Benjamin Goh, Apollos C. Kim, and Lynne M. Quarmby. "The FA2 gene of Chlamydomonas encodes a NIMA family kinase with roles in cell cycle progression and microtubule severing during deflagellation." Journal of Cell Science 115, no. 8 (April 15, 2002): 1759–68. http://dx.doi.org/10.1242/jcs.115.8.1759.
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The NIMA kinases are one of several families of kinases that participate in driving the eukaryotic cell cycle. NIMA-related kinases have been implicated in G2/M progression, chromatin condensation and regulation of the centrosome cycle. Here we report the identification of a new member of this family, FA2, from Chlamydomonas reinhardtii. FA2 was originally discovered in a genetic screen for deflagellation-defective mutants. We have previously shown that FA2 is essential for basal-body/centriole-associated microtubule severing. We now report that the FA2 NIMA-related kinase also plays a role in cell cycle progression in Chlamydomonas. This is the first indication that members of the NIMA family might exert their effects through the regulation of microtubule severing.
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Palazzo, A. F. "Localized Stabilization of Microtubules by Integrin- and FAK-Facilitated Rho Signaling." Science 303, no. 5659 (February 6, 2004): 836–39. http://dx.doi.org/10.1126/science.1091325.
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Xie, Zhigang, and Li-Huei Tsai. "Cdk5 Phosphorylation of FAK Regulates Centrosome-Associated Microtubules and Neuronal Migration." Cell Cycle 3, no. 2 (February 2004): 105–8. http://dx.doi.org/10.4161/cc.3.2.646.
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Wei, Wei-Chun, Hsi-Hui Lin, Meng-Ru Shen, and Ming-Jer Tang. "Mechanosensing machinery for cells under low substratum rigidity." American Journal of Physiology-Cell Physiology 295, no. 6 (December 2008): C1579—C1589. http://dx.doi.org/10.1152/ajpcell.00223.2008.
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Mechanical stimuli are essential during development and tumorigenesis. However, how cells sense their physical environment under low rigidity is still unknown. Here we show that low rigidity of collagen gel downregulates β1-integrin activation, clustering, and focal adhesion kinase (FAK) Y397 phosphorylation, which is mediated by delayed raft formation. Moreover, overexpression of autoclustered β1-integrin (V737N), but not constitutively active β1-integrin (G429N), rescues FAKY397 phosphorylation level suppressed by low substratum rigidity. Using fluorescence resonance energy transfer to assess β1-integrin clustering, we have found that substratum rigidity between 58 and 386 Pa triggers β1-integrin clustering in a dose-dependent manner, which is highly dependent on actin filaments but not microtubules. Furthermore, augmentation of β1-integrin clustering enhances the interaction between β1-integrin, FAK, and talin. Our results indicate that contact with collagen fibrils is not sufficient for integrin activation. However, substratum rigidity is required for integrin clustering and activation. Together, our findings provide new insight into the mechanosensing machinery and the mode of action for epithelial cells in response to their physical environment under low rigidity.
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Hegmann, T. E., J. L. Lin, and J. J. Lin. "Probing the role of nonmuscle tropomyosin isoforms in intracellular granule movement by microinjection of monoclonal antibodies." Journal of Cell Biology 109, no. 3 (September 1, 1989): 1141–52. http://dx.doi.org/10.1083/jcb.109.3.1141.
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Chicken embryo fibroblast (CEF) cells were microinjected with several different monoclonal antibodies that recognize certain nonmuscle isoforms of tropomyosin. Immediately after injection, cells were recorded with a time-lapse video imaging system; later analysis of the tapes revealed that particles in cells injected with one of these antibodies (CG1, specific for CEF tropomyosin isoforms 1 and 3) showed a dramatic decrease in instantaneous speed while moving, distance moved per saltation, and proportion of time spent in motion. Injection of Fab fragments of CG1 resulted in similar changes in the pattern of granule movement. This inhibition of granule movement by CG1 antibody was reversible; at 2.5 h after injection, granules in injected cells had already reached three-fourths of normal speed. The speed of granule movement in cells injected either with antibody specific for tropomyosin isoforms not present in CEF cells, or with CG1 antibody preabsorbed with tropomyosin, was not significantly different from the speed of granules in uninjected cells. When cells were injected with CG1 or Fab fragments of CG1, fixed, and counter-stained with rabbit antibodies to reveal the microtubule, microfilament, and intermediate filament systems, no obvious differences from the patterns normally seen in uninjected cells were observed. Examination of the ultrastructure of injected cells by EM confirmed the presence of apparently intact and normal microtubule, actin, and intermediate filament networks. These experiments suggest that tropomyosin may play an important role in the movement of vesicles and organelles in the cell cytoplasm. Also, we have shown previously that the CG1 determinant can undergo a motility-dependent change in reactivity, that may be important for the regulatory function of nonmuscle tropomyosin (Hegmann, T. E., J. L.-C. Lin, and J. J.-C. Lin. 1988. J. Cell Biol. 106:385-393). Therefore, in addition to postulated microtubule-based motors, microfilaments may play a critical role in regulating granule movement in nonmuscle cells.
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Smyrek, I., B. Mathew, S. C. Fischer, S. M. Lissek, S. Becker, and E. H. K. Stelzer. "E-cadherin, actin, microtubules and FAK dominate different spheroid formation phases and important elements of tissue integrity." Biology Open 8, no. 1 (December 21, 2018): bio037051. http://dx.doi.org/10.1242/bio.037051.
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Granic, Antoneta, Jaya Padmanabhan, Michelle Norden, and Huntington Potter. "Alzheimer Aβ Peptide Induces Chromosome Mis-Segregation and Aneuploidy, Including Trisomy 21: Requirement for Tau and APP." Molecular Biology of the Cell 21, no. 4 (February 15, 2010): 511–20. http://dx.doi.org/10.1091/mbc.e09-10-0850.
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Both sporadic and familial Alzheimer's disease (AD) patients exhibit increased chromosome aneuploidy, particularly trisomy 21, in neurons and other cells. Significantly, trisomy 21/Down syndrome patients develop early onset AD pathology. We investigated the mechanism underlying mosaic chromosome aneuploidy in AD and report that FAD mutations in the Alzheimer Amyloid Precursor Protein gene, APP, induce chromosome mis-segregation and aneuploidy in transgenic mice and in transfected cells. Furthermore, adding synthetic Aβ peptide, the pathogenic product of APP, to cultured cells causes rapid and robust chromosome mis-segregation leading to aneuploid, including trisomy 21, daughters, which is prevented by LiCl addition or Ca2+ chelation and is replicated in tau KO cells, implicating GSK-3β, calpain, and Tau-dependent microtubule transport in the aneugenic activity of Aβ. Furthermore, APP KO cells are resistant to the aneugenic activity of Aβ, as they have been shown previously to be resistant to Aβ-induced tau phosphorylation and cell toxicity. These results indicate that Aβ-induced microtubule dysfunction leads to aneuploid neurons and may thereby contribute to the pathogenesis of AD.
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You, Bang-Jau, Yang-Chang Wu, Bo-Ying Bao, Chi-Yu Wu, Ya-Win Yang, Yu-Hao Chang, and Hong-Zin Lee. "Rottlerin InhibitsLonicera japonica-Induced Photokilling in Human Lung Cancer Cells through Cytoskeleton-Related Signaling Cascade." Evidence-Based Complementary and Alternative Medicine 2011 (2011): 1–9. http://dx.doi.org/10.1155/2011/193842.
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This study demonstrated that many apoptotic signaling pathways, such as Rho family, PKC family, MAP kinase family, and mitochondria-mediated apoptotic pathway, were triggered byLonicera japonicaextracts and irradiation in CH27 cells. Rottlerin, a PKCδ-selective inhibitor, reversed the photoactivatedLonicera japonicaextract-induced decrease in PKCδprotein expression and change in cell morphology in this study. In addition, rottlerin inhibited the photoactivatedLonicera japonica-induced decrease in protein expression of Ras, ERK, p38, PKCα, and PKCε, which are the kinases of prosurvival signaling pathway. We also demonstrated that pretreatment with rottlerin prevented actin microfilaments and microtubules from damage during the photoactivatedLonicera japonica-induced CH27 cell death. Furthermore, the promotion of the cytoskeleton-related signaling cascade following rottlerin by upregulation of cytoskeleton-related mediators (p38, HSP27, FAK, paxillin, and tubulin) and molecules of downstream of F-actin (mitochondria-mediated apoptosis pathway) reduces CH27 cell death, indicating that cytoskeleton is the potential target in the photoactivatedLonicera japonicaextract-induced photokilling of CH27 cells.
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Wu, S., S. Chasalow, H. Lee, L. Xu, B. Paul, O. Mokliatchouk, W. F. Symmans, K. E. Zerba, L. Pusztai, and E. Clark. "Identification of predictive markers to differentiate ixabepilone from paclitaxel activity in ER-negative breast cancer patients." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 2525. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.2525.
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2525 Background: Ixabepilone (BMS-247550) is a microtubule stabilizing agent with demonstrable therapeutic value in taxane- refractory breast cancer (BC) patients. Biomarkers to predict either ixabepilone or paclitaxel activity in BC patients have previously been reported. However, markers that differentiate response to the two agents have yet to be identified. This study sought to discover predictive markers that will enable patient selection to differentially enhance response to ixabepilone or paclitaxel in ER-negative (ER-) patients. Materials and Methodologies: Pre-treatment gene expression profiles were generated for 62 ER- patients treated with ixabepilone in clinical study CA163080, and 51 ER- patients treated with T/FAC (paclitaxel and fluorouracil-doxorubicin-cyclophosphamide) in clinical study MDA133. Biomarkers differentially predictive of complete pathological response in breast were identified through gene set enrichment analysis (GSEA) or classification by threshold gradient descent (TGD). Gene knockdown by siRNA was used to study some of these candidate markers. Results: Four candidate models that differentiate response to ixabepilone treatment and taxane-containing therapy were identified. Two of the models, found by GSEA, are based on expression levels for single microtubule-related genes: transforming, acidic coiled-coil containing protein 3 (TACC3) and chromosome condensation protein G (HCAP-G). The potential of HCAP-G as a differential marker was supported by siRNA studies. Two of the models, found by TGD, are based on expression levels for 26 and 20 genes. Areas under the ROC curves for the models applied to each study separately are given in the table . Conclusions: We have identified four predictive models that differentiate response in a clinical trial of ixabepilone from that in a trial of T/FAC. A clinical trial is under way to further evaluate their utility for differentiating response to ixabepilone- and taxane-containing regimens. [Table: see text] [Table: see text]
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Lunn, J. Adrian, and Enrique Rozengurt. "M1600 Focal Adhesion Kinase (FAK) Plays a Critical Role in Early Cell Migration By Affecting the Microtubular Cytoskeleton." Gastroenterology 136, no. 5 (May 2009): A—392. http://dx.doi.org/10.1016/s0016-5085(09)61800-x.
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Powell, Richard D., Vishwas N. Joshi, Carol M. R. Halsey, James F. Hainfeld, Gerhard W. Hacker, Cornelia Hauser-Kronberger, Wolfgang H. Muss, and Peter M. Takvorian. "Combined Cy3 / Nanogold Conjugates for ImmunocytoChemistry and in Situ Hybridization." Microscopy and Microanalysis 5, S2 (August 1999): 478–79. http://dx.doi.org/10.1017/s1431927600015713.
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Fluorescein and the 1.4 nm Nanogold® gold cluster label may be incorporated into a single Fab’ immunoprobe by separate cross-linking reactions, to give a probe which labels antigenic sites in a single step for correlative fluorescence and electron microscope visualization. These probes show high labeling density, labeling a pre-mRNA splicing factor in the HeLa cell nucleus; Microtubules were also densely labeled using fluorescence, other optical modalities, and electron microscopy; in a parallel experiment, a 5 nm colloidal gold probe gave only occasional labeling. We now describe Fab’ and streptavidin probes containing both Nanogold® and the fluorescent cyanine dye, Cy3.F(ab’)2 Goat anti-Mouse IgG and F(ab’)2 goat anti-rabbit IgG fragments were reductively cleaved to Fab’ fragments using dithiothreitol (DTT) or mercaptoethylamine hydrochloride (MEA), which selectively reduce the F(ab’)2 hinge disulfide bonds, with 5 mm EDTA to prevent reoxidation. Fab’ fragments were isolated by gel filtration (coarse gel: GH25, Amicon) then labeled with Monomaleimido- Nanogold® which reacts site-specifically with thiols. Streptavidin was labeled using Mono- Sulfo-NHS-Nanogold® at pH 7.5. Nanogold® conjugates were isolated by gel filtration (Superose-12 column, Pharmacia), then reacted with excess Cy3 monofunctional NHS ester (labeling kit, Amersham Life Sciences) at pH 7.5; dual-labeled conjugates were isolated by gel filtration (Superose-12).
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Soppina, Virupakshi, Jeffrey F. Herbstman, Georgios Skiniotis, and Kristen J. Verhey. "Luminal Localization of α-tubulin K40 Acetylation by Cryo-EM Analysis of Fab-Labeled Microtubules." PLoS ONE 7, no. 10 (October 26, 2012): e48204. http://dx.doi.org/10.1371/journal.pone.0048204.
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Robinson, John M., and Dale D. Vandré. "Efficient Immunocytochemical Labeling of Leukocyte Microtubules with FluoroNanogold: An Important Tool for Correlative Microscopy." Journal of Histochemistry & Cytochemistry 45, no. 5 (May 1997): 631–42. http://dx.doi.org/10.1177/002215549704500501.
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We tested the immunoprobe FluoroNanogold (FNG) for its utility as an immunocytochemical labeling reagent. This immunoprobe consists of a 1.4-nm gold particle to which a specific Fab' fragment and a fluorochrome are conjugated. We employed the microtubules (MTs) of human phagocytic leukocytes as a model system for testing the usefulness of FNG as a secondary antibody for immunocytochemistry. We show that these fluorescently labeled ultrasmall immunogold particles are very efficient for labeling MTs in these cells. The signal from FNG can be detected directly by fluorescence microscopy or indirectly by other modes of optical microscopy and electron microscopy, after silver-enhancement of the gold. The spatial resolution of immunolabeled MTs obtained with FNG and silver enhancement was comparable to that of conventional immunofluorescence detection. Colloidal gold (5-nm and 10-nm in diameter), on the other hand, failed to label MTs in cells prepared in a similar manner. This difference in labeling was due in large part to greater penetration of 1.4-nm gold into aldehyde-fixed cells than either 5-nm or 10-nm gold particles. The fluorescent 1.4-nm immunoprobe was shown to be an important new tool for general use in correlative microscopy.
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Pusztai, L., F. Andre, C. Mazouni, C. Liedtke, S. Kau, D. Frye, M. Green, A. M. Gonzalez-Angulo, W. F. Symmans, and G. N. Hortobagyi. "HER2 expression and efficacy of preoperative paclitaxel and 5-fluorouracil, cyclophosphamide, doxorubicin chemotherapy in breast cancer." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 548. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.548.
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548 Purpose: We examined the correlation between HER2 expression and pathologic complete response (pCR) to paclitaxel/FAC (T/FAC) preoperative chemotherapy in stage II-III breast cancer. Patients and Methods: Retrospective analysis of data including 534 patients treated with preoperative T/FAC was performed. Gene expression results were available from a subset of 132 patients and were used to examine the co-expression of HER2 and topoisomerase II a (TOP2A) and microtubule associated protein tau (MAP-Tau). Results: Of the 534 patients, 105 (20%) had HER2 overexpressing (HER2+) breast cancer. The pCR rates were 33% and 15% for patients with HER2+ and HER2- tumors (P<0.001). The 5-year relapse-free survival rates were 94% and 70% in HER2+ tumors with and without pCR (P=0.009). In multivariate analysis ER-negative status (OR 3.1, 95% CI 1.7–5.5, P < 0.001), high nuclear grade (OR 6.7, 95% CI 3.1–14.2, P < 0.001), weekly paclitaxel regimen (OR 2.6, 95% CI 1.5–4.5, P <0.001), and HER2 overexpression (OR 2.4, 95% CI 1.3–4.2, P = 0.003) were significant predictors of pCR. Among ER- cancers, the pCR rates were 50% (95%CI: 36–64%) in HER2+ (n=24/48) and 30% (95%CI: 23–37%) in HER2- disease (n=48/159), P = 0.02. Among ER+ cancers, the pCR rate was 19% (95% CI: 9–29%) in HER2+ (n=11/57) and 6% (95% CI: 3–9%) in HER2- disease (n=17/270), P = 0.003. In the gene expression data, HER2 overexpression was associated with lower expression of MAP-tau (mean 303 vs 500, p=0.001) and higher expression of TOP2A mRNAs (mean 398 vs 274, p=0.048) in patients with ER+ disease. All ER- cancers had low MAP-Tau expression (compared to ER+ cancers) regardless of HER2 status. Conclusion: HER2 overexpression is associated with higher rate of pCR to preoperative T/FAC chemotherapy in both ER- and ER+ cancers. HER2 overexpression correlates with increased TOP2A and decreased MAP-Tau expression in ER+ cancers which could explain increased anthracycline and taxane sensitivity of these tumors. No significant financial relationships to disclose.
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Goubard, Armelle, Martine Humbert, Colin Mansfield, Olivier Hermine, Patrice Dubreuil, and The AB8939 Study Group. "AB8939, a Microtubule-Destabilizing Agent with Potential to Overcome Multidrug Resistance, Is Active across the Range (M0-M7) of Acute Myeloid Leukemia Subtypes." Blood 134, Supplement_1 (November 13, 2019): 5154. http://dx.doi.org/10.1182/blood-2019-127021.
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Compound AB8939 is a structurally novel, small chemical molecule, synthesized tubulin inhibitor that can circumvent two of the major resistance mechanisms in acute myeloid leukemia (AML), namely P-glycoprotein (Pgp) and myeloperoxidase (MPO) mediated resistance, thereby conferring an important advantage over traditional tubulin inhibitors. A series of ex vivo and in vivo studies provide proof-of-concept that AB8939 has broad anti-proliferative activity across the breath of acute myeloid leukemia (AML) subtypes, i.e. M0 through M7 of the French-American-British AML classification. Acute myeloid leukemia blasts were isolated from patients' peripheral blood and/or bone marrow samples, collected either at the time of diagnosis or following relapse, and also from patient derived xenograft (PDX) models. After purification, mononuclear cells were treated for 48 hours with various concentrations of AB8939 or cytarabine (Ara-C) and analyzed in a cell proliferation/viability assay. AB8939 produced a strong anti-proliferative action against blasts isolated from AML patients with a majority of IC50 values ranging from 1.4 nM to 1.0 µM. Two-thirds of AML patients had nanomolar sensitivity to AB8939 (IC50 ≤ 500 nM), while 44% where very sensitive (IC50 ≤ 100 nM) and 11% were highly sensitive (IC50 ≤ 10 nM). The potential of AB8939 to overcome Ara-C-resistance was also evident with 66% of Ara-C-resistant blasts (i.e. IC50 >5 µM) being sensitive to AB8939. Notably, AB8939 demonstrated activity across the entire spectrum of AML subtypes, according to the French-American-British (FAB) AML classification, with an IC50 of < 50 nM in M0, M1, M4, M5 and M6 subtypes, corresponding to over 90% of the AML patient population. A slightly lower sensitivity was observed for the M3 subtype (IC50 = 1.25 µM). Additionally, potent AB8939 activity was also seen in Ara-C-insensitive biphenotypic and mixed-phenotype acute leukemia samples. All FAB categories other than M7 were tested in the abovementioned ex vivo assessment. Acute megakaryocytic leukemia (FAB AML subtype M7) is a rare form of adult AML, accounting for only 1% of cases, and is associated with resistance to standard treatment and poor prognosis. The effect of AB8939 in this subtype was assessed in vivo using an AMKL26 model, an NSG mouse model based on cells isolated from a patient with an aggressive acute megakaryocytic leukemia presenting the ETO2-GLIS2 fusion oncogene. Following post graft detection of blasts, single agent AB8939 was administered intravenously at a dose of 2 mg/kg for three consecutive days per week (3 ON / 4 OFF) for 2 weeks and then at 5 mg/kg for three consecutive days per week (3 ON / 4 OFF) for 1 week. At the end of the 3-week treatment period, blast detection in bone marrow was performed via bioluminescence imaging with comparison to vehicle-treated controls. As seen in the figure below, single agent AB8939 showed strong anti-leukemic activity in this AMKL26 mouse model as evidenced by the near eradication of blasts. No blasts could be detected in 6 out of 8 mice treated with AB8939. At the described dosing schedule, AB8939 was well-tolerated with no toxicity-related deaths and no impact on animal body weight or behavior. These findings provide preclinical proof of concept for the development of AB8939 as a next-generation tubulin inhibitor for AML, in particular for poor-prognosis AML subsets and relapse/refractory AML; i.e. patients that currently have very limited therapeutic options and represent the highest unmet medical need. Disclosures Goubard: AB Science: Employment. Humbert:AB Science: Employment. Mansfield:AB Science: Employment, Patents & Royalties. Hermine:AB Science: Membership on an entity's Board of Directors or advisory committees. AB8939 Study Group:AB Science: Consultancy, Employment.
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Dou, Xiaoqian, Qinzhi Xu, Bo Dong, Guili Xu, Niliang Qian, Cuima Yang, Hongjie Li, Liting Chen, Xin Gao, and Haifeng Song. "Anti-c-MET Fab-Grb2-Gab1 Fusion Protein-Mediated Interference of c-MET Signaling Pathway Induces Methuosis in Tumor Cells." International Journal of Molecular Sciences 23, no. 19 (October 10, 2022): 12018. http://dx.doi.org/10.3390/ijms231912018.
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Bio-macromolecules have potential applications in cancer treatment due to their high selectivity and efficiency in hitting therapeutic targets. However, poor cell membrane permeability has limited their broad-spectrum application in cancer treatment. The current study developed highly internalizable anti-c-MET antibody Fab fusion proteins with intracellular epitope peptide chimera to achieve the dual intervention from the extracellular to intracellular targets in tumor therapy. In vitro experiments demonstrated that the fusion proteins could interfere with the disease-associated intracellular signaling pathways and inhibit the uncontrolled proliferation of tumor cells. Importantly, investigation of the underlying mechanism revealed that these protein chimeras could induce vacuolation in treated cells, thus interfering with the normal extension and arrangement of microtubules as well as the mitosis, leading to the induction of methuosis-mediated cell death. Furthermore, in vivo tumor models indicated that certain doses of fusion proteins could inhibit the A549 xenograft tumors in NOD SCID mice. This study thus provides new ideas for the intracellular delivery of bio-macromolecules and the dual intervention against tumor cell signaling pathways.
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Cehlar, Ondrej, Rostislav Skrabana, Andrej Kovac, Branislav Kovacech, and Michal Novak. "Crystallization and preliminary X-ray diffraction analysis of tau protein microtubule-binding motifs in complex with Tau5 and DC25 antibody Fab fragments." Acta Crystallographica Section F Structural Biology and Crystallization Communications 68, no. 10 (September 25, 2012): 1181–85. http://dx.doi.org/10.1107/s1744309112030382.
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Barroso, M., and ES Sztul. "Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome." Journal of Cell Biology 124, no. 1 (January 1, 1994): 83–100. http://dx.doi.org/10.1083/jcb.124.1.83.
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We have used temperature and nocodazole blocks in an in vivo basolateral to apical transcytosis assay to dissociate the early transcytotic steps occurring during the formation of transcytotic vesicles and their microtubule-dependent translocation into the apical region, from the late steps when transcytotic cargo is delivered into the apical media. We found that polarized MDCK cells transfected with rabbit polymeric IgA receptor (pIgA-R) internalize basolaterally added pIgA-R ligand ([Fab]2 fragment of IgG against the receptor's ectodomain) at 17 degrees C but do not deliver it to the apical PM. Instead, the ligand accumulates in an apically localized transcytotic compartment, distal to the basolateral endosome and the microtubule-requiring translocation step. We have characterized this compartment and show that it is distinct from basolateral transferrin recycling endosomes, basolateral early endosomes or late endosomes or lysosomes. The apical transcytotic compartment colocalizes with the compartment containing apically recycling membrane markers (ricin and apically internalized pIgA-R ligand) but is distinct from the compartment receiving apically internalized fluid phase marker (BSA). This compartment is an intermediate station of the overall pathway since transcytotic ligand can exit the compartment and be released into the apical medium when cells preloaded at 17 degrees C are subsequently incubated at 37 degrees C. We have used this system to examine the effect of Brefeldin A (BFA) and the involvement of trimeric GTPases in the late (post apical transcytotic compartment) steps of the transcytotic pathway. We found that addition of BFA or cholera toxin, a known activator of Gs alpha, to cells preloaded with transcytotic ligand at 17 degrees C significantly inhibits the exit of ligand from the apical transcytotic compartment. General structure and function of the apical endosome are not affected since neither BFA nor cholera toxin inhibit the recycling of apically internalized membrane markers (ricin and pIgA-R ligand) from the same compartment. The data suggest that transcytosis connects the "membrane-sorting" sub-domain of the basolateral endosome with a homologous sub-domain of the apical endosome and that exit of transcytosing cargo from the apical endosome is controlled by a BFA and trimeric G protein sensitive mechanism, distinct from that used for recycling of apically internalized proteins (ricin or pIgA-R).
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Alzahrani, Alanoud Bakheet. "Gene Expression in Proliferative Diabetic Retinopathy Using RNA-Seq Data: A Computational Study on Saudi Patients." Bioscience Biotechnology Research Communications 14, no. 4 (December 25, 2021): 1760–63. http://dx.doi.org/10.21786/bbrc/14.4.56.
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Proliferative diabetic retinopathy is the widespread type of DM which causes chronic as well as progressive alterations at microvascular level, which particularly effects the eye. The main characteristic of this disease is the development of few new blood vessels around the retina of eye as well as at the posterior region of eye segments. For our computational analysis 155 differentially expressed genes calculated through paired t test statistics analysis using the GenePattern platform, of proliferative diabetic retinopathy in Saudi patients were downloaded. Among the 155 genes, 95 were upregulated, and 60 were downregulated. The Annotation Cluster (FAC) tool in the (DAVID) (http://david.abcc.ncifcrf.gov/home.jsp) was used to identify biological processes that are abundant in proliferative diabetic retinopathy (PDR). The functions required for response to mRNA splicing, intracellular protein transport, mRNA processing, microtubule cytoskeleton structure, and atrioventricular canal formation are represented by the GO keywords that are abundant in genes. We used the KAAS web server to identify the biological pathways of these DEGs in addition to DAVID functional analysis and found that the majority of the DEGs were associated with important biological processes, with many being classified in metabolic pathways, Spliceosome, Cell cycle, or being involved in the mRNA surveillance pathway. findings are consistent with those of earlier research. To corroborate the predictions stated in this work, which will demonstrate the role enhanced functional processes, experimental validation will be necessary.
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RUBIO, Mercedes POZUELO, Kathryn M. GERAGHTY, Barry H. C. WONG, Nicola T. WOOD, David G. CAMPBELL, Nick MORRICE, and Carol MACKINTOSH. "14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking." Biochemical Journal 379, no. 2 (April 15, 2004): 395–408. http://dx.doi.org/10.1042/bj20031797.
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14-3-3-interacting proteins were isolated from extracts of proliferating HeLa cells using 14-3-3 affinity chromatography, eluting with a phosphopeptide that competes with targets for 14-3-3 binding. The isolated proteins did not bind to 14-3-3 proteins (14-3-3s) after dephosphorylation with protein phosphatase 2A (PP2A), indicating that binding to 14-3-3s requires their phosphorylation. The binding proteins identified by tryptic mass fingerprinting and Western blotting include many enzymes involved in generating precursors such as purines (AMP, GMP and ATP), FAD, NADPH, cysteine and S-adenosylmethionine, which are needed for cell growth, regulators of cell proliferation, including enzymes of DNA replication, proteins of anti-oxidative metabolism, regulators of actin dynamics and cellular trafficking, and proteins whose deregulation has been implicated in cancers, diabetes, Parkinsonism and other neurological diseases. Several proteins bound to 14-3-3–Sepharose in extracts of proliferating cells, but not in non-proliferating, serum-starved cells, including a novel microtubule-interacting protein ELP95 (EMAP-like protein of 95 kDa) and a small HVA22/Yop1p-related protein. In contrast, the interactions of 14-3-3s with the N-methyl-d-aspartate receptor 2A subunit and NuMA (nuclear mitotic apparatus protein) were not regulated by serum. Overall, our findings suggest that 14-3-3s may be central to integrating the regulation of biosynthetic metabolism, cell proliferation, survival, and other processes in human cells.
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Apodaca, G., L. A. Katz, and K. E. Mostov. "Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes." Journal of Cell Biology 125, no. 1 (April 1, 1994): 67–86. http://dx.doi.org/10.1083/jcb.125.1.67.
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Classically, the polymeric immunoglobulin receptor and its ligand, IgA, are thought to be sorted from basolateral early endosomes into transcytotic vesicles that directly fuse with the apical plasma membrane. In contrast, we have found that in MDCK cells IgA is delivered from basolateral endosomes to apical endosomes and only then to the apical cell surface. When internalized from the basolateral surface of MDCK cells IgA is found to accumulate under the apical plasma membrane in a compartment that is accessible to two apically added membrane markers: anti-secretory component Fab fragments, and avidin internalized from the biotinylated apical pole of the cell. This accumulation occurs in the presence of apical trypsin, which prevents internalization of the ligand from the apical cell surface. Using a modification of the diaminobenzidine density-shift assay, we estimate that approximately 80% of basolaterally internalized IgA resides in the apical endosomal compartment. In addition, approximately 50% of basolaterally internalized transferrin, a basolateral recycling protein, has access to this apical endosomal compartment and is efficiently recycled back to the basolateral surface. Microtubules are required for the organization of the apical endosomal compartment and it is dispersed in nocodazole-treated cells. Moreover, this compartment is largely inaccessible to fluid-phase markers added to either pole of the cell, and therefore seems analogous to the recycling endosome described in nonpolarized cells. We propose a model in which transcytosis is not a specialized pathway that uses unique transcytotic vesicles, but rather combines portions of pathways used by non-transcytosing molecules.
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Gray, Elizabeth, Kelly Hensley, Sean Allred, Esther Trueblood, John Gosink, Robert Thurman, Kaleb Smith, et al. "617 Tisotumab vedotin shows immunomodulatory activity through induction of immunogenic cell death." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A653. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0617.
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BackgroundTisotumab vedotin (TV) is an investigational antibody-drug conjugate composed of a tissue factor (TF)-directed human monoclonal antibody covalently linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable linker. TV demonstrated single agent activity (24% objective response rate [ORR]) in previously treated recurrent or metastatic cervical cancer (NCT03438396) where currently, there is no standard of care and ORRs are typically less than 15% and often of limited duration.1–8 TV is currently being evaluated in combination with pembrolizumab (PD-1 inhibitor), bevacizumab, or carboplatin in cervical cancer (NCT03786081), or as a monotherapy in multiple other solid tumors (NCT03913741, NCT03485209, NCT03657043). The anti-tumor activity of TV may be multimodal as TV can induce tumor cell death through several mechanisms, including direct and bystander MMAE-mediated cytotoxicity, as well as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and immunogenic cell death (ICD)].9 10 To better characterize immune-mediated tumor cell killing by TV and further the rationale for combination with pembrolizumab, we set out to refine our understanding of TV-mediated ICD and subsequent immunomodulatory effects.MethodsWe evaluated the ability of TV to mediate activation of immune cells in vitro using co-cultures of TF-expressing tumor cells and human peripheral blood mononuclear cells (PBMCs). We also assessed the ability of TV to induce recruitment of innate immune cells to tumors in vivo using a TF-expressing xenograft tumor model.ResultsIn vitro, tumor cells treated with TV showed several hallmarks of immunogenic cell death, including markers of endoplasmic reticulum (ER) stress and release of ATP and high mobility group protein B1 (HMGB1). Co-culture of TV-killed tumor cells with allogeneic human PBMCs led to innate immune cell activation (measured by upregulation of the costimulatory molecule CD86) and T cell proliferation. Combination with PD-1 blockade further amplified the immune response, leading to enhanced T cell proliferation and cytokine production. Moreover, in vivo studies demonstrated that TV treatment led to recruitment of F4/80+ and CD11c+ innate immune cells to xenograft tumors.ConclusionsThese data show that, in preclinical models, TV induces immunogenic tumor cell death, which can promote activation and recruitment of immune cells to the tumor. The totality of in vitro and in vivo data provides evidence for the immunomodulatory effects of TV and bolsters rationale for combining TV with immune checkpoint agents. Ongoing analyses aim at further characterizing the immune response induced by TV in preclinical models and patients.AcknowledgementsWe would like to thank Kristen Gahnberg for embedding and sectioning tissues for these studies and Anthony Cao for his early work identifying hallmarks of ICD in response to TV treatment in vitro.9Ethics ApprovalAnimals studies were approved by and conducted in accordance with Seattle Genetics Institutional Care and Use Committee protocol #SGE-029.ConsentN/AReferencesVan den berg YW, Osanto S, Reitsma PH, Versteeg HH. The relationship between tissue factor and cancer progression: insights from bench and bedside. Blood 2012;119(4):924–932.Miller DS, Blessing JA, Bodurka DC, Bonebrake AJ, Schorge JO. Evaluation of pemetrexed (Alimta, LY231514) as second line chemotherapy in persistent or recurrent carcinoma of the cervix: a phase II study of the Gynecologic Oncology Group. Gynecol Oncol 2008;110(1):65–70.Bookman MA, Blessing JA, Hanjani P, Herzog TJ, Andersen WA. Topotecan in squamous cell carcinoma of the cervix: A Phase II study of the gynecologic oncology group. Gynecol Oncol 2000;77(3):446–449.Garcia AA, Blessing JA, Vaccarello L, Roman LD. Phase II clinical trial of docetaxel in refractory squamous cell carcinoma of the cervix: a Gynecologic Oncology Group Study. Am J Clin Oncol 2007;30(4):428–431.Monk BJ, Sill MW, Burger RA, Gray HJ, Buekers TE, Roman LD. Phase II trial of bevacizumab in the treatment of persistent or recurrent squamous cell carcinoma of the cervix: a gynecologic oncology group study. J Clin Oncol 2009;27(7):1069–1074.Santin AD, Sill MW, Mcmeekin DS, et al. Phase II trial of cetuximab in the treatment of persistent or recurrent squamous or non-squamous cell carcinoma of the cervix: a gynecologic oncology group study. Gynecol Oncol 2011;122(3):495–500.Schilder RJ, Blessing J, Cohn DE. Evaluation of gemcitabine in previously treated patients with non-squamous cell carcinoma of the cervix: a phase II study of the Gynecologic Oncology Group. Gynecol Oncol 2005;96(1):103–107.Chung HC, Ros W, Delord JP, et al. Efficacy and safety of pembrolizumab in previously treated advanced cervical cancer: results from the phase II KEYNOTE-158 study. J Clin Oncol 2019;37(17):1470–1478.Alley SC, Harris JR, Cao A, et al. Tisotumab vedotin induces antitumor activity through MMAE-Mediated, Fc-Mediated, and Fab-Mediated Effector Functions In Vitro [abstract]. Cancer Res. 2019;79(13 Suppl):Abstract 221.Breij E, de Goeij B, Verploegen S, et al. An antibody-drug conjugate that targets tissue factor exhibits potent therapeutic activity against a broad range of solid tumors. Cancer Res 2014 ;74(4):1214–1226.
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Li, Huitao, Shiwen Liu, Siwen Wu, Renshan Ge, and C. Yan Cheng. "NC1-Peptide From Collagen α3 (IV) Chains in the Basement Membrane of Testes Regulates Spermatogenesis via p-FAK-Y407." Endocrinology 161, no. 10 (August 6, 2020). http://dx.doi.org/10.1210/endocr/bqaa133.
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Abstract The blood–testis barrier (BTB) in the testis is an important ultrastructure to support spermatogenesis. This blood-tissue barrier undergoes remodeling at late stage VII to early stage IX of the epithelial cycle to support the transport of preleptotene spermatocytes across the BTB to prepare for meiosis I/II at the apical compartment through a mechanism that remains to be delineated. Studies have shown that NC1-peptide-derived collagen α3 (IV) chain in the basement membrane is a bioactive peptide that induces BTB remodeling. It also promotes the release of fully developed spermatids into the tubule lumen. Thus, this endogenously produced peptide coordinates these 2 cellular events across the seminiferous epithelium. Using an NC1-peptide complementary deoxyribonucleic acid (cDNA) construct to transfect adult rat testes for overexpression, NC1-peptide was found to effectively induce germ cell exfoliation and BTB remodeling, which was associated with a surge and activation of p-rpS6, the downstream signaling protein of mTORC1 and the concomitant downregulation of p-FAK-Y407 in the testis. In order to define the functional relationship between p-rpS6 and p-FAK-Y407 signaling to confer the ability of NC1-peptide to regulate testis function, a phosphomimetic (and thus constitutively active) mutant of p-FAK-Y407 (p-FAK-Y407E-MT) was used for its co-transfection, utilizing Sertoli cells cultured in vitro with a functional tight junction (TJ) barrier that mimicked the BTB in vivo. Overexpression of p-FAK-Y407E-MT blocked the effects of NC1-peptide to perturb Sertoli cell BTB function by promoting F-actin and microtubule cytoskeleton function, and downregulated the NC1-peptide-mediated induction of p-rpS6 activation. In brief, NC1-peptide is an important endogenously produced biomolecule that regulates BTB dynamics.
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Li, Yang, Kai-Xuan Li, Wei-Lin Hu, David M. Ojcius, Jia-Qi Fang, Shi-Jun Li, Xu'ai Lin, and Jie Yan. "Endocytic recycling and vesicular transport systems mediate transcytosis of Leptospira interrogans across cell monolayer." eLife 8 (April 23, 2019). http://dx.doi.org/10.7554/elife.44594.
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Many bacterial pathogens can cause septicemia and spread from the bloodstream into internal organs. During leptospirosis, individuals are infected by contact with Leptospira-containing animal urine-contaminated water. The spirochetes invade internal organs after septicemia to cause disease aggravation, but the mechanism of leptospiral excretion and spreading remains unknown. Here, we demonstrated that Leptospira interrogans entered human/mouse endothelial and epithelial cells and fibroblasts by caveolae/integrin-β1-PI3K/FAK-mediated microfilament-dependent endocytosis to form Leptospira (Lep)-vesicles that did not fuse with lysosomes. Lep-vesicles recruited Rab5/Rab11 and Sec/Exo-SNARE proteins in endocytic recycling and vesicular transport systems for intracellular transport and release by SNARE-complex/FAK-mediated microfilament/microtubule-dependent exocytosis. Both intracellular leptospires and infected cells maintained their viability. Leptospiral propagation was only observed in mouse fibroblasts. Our study revealed that L. interrogans utilizes endocytic recycling and vesicular transport systems for transcytosis across endothelial or epithelial barrier in blood vessels or renal tubules, which contributes to spreading in vivo and transmission of leptospirosis.
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McGrail, Daniel J., Niti N. Khambhati, Mark X. Qi, Krishan S. Patel, Nithin Ravikumar, Chandler P. Brandenburg, and Michelle R. Dawson. "Alterations in Ovarian Cancer Cell Adhesion Drive Taxol Resistance by Increasing Microtubule Dynamics in a FAK-dependent Manner." Scientific Reports 5, no. 1 (April 17, 2015). http://dx.doi.org/10.1038/srep09529.
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