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1
Bryce, Nicole S., Galina Schevzov, Vicki Ferguson, Justin M. Percival, Jim J. C. Lin, Fumio Matsumura, James R. Bamburg, et al. "Specification of Actin Filament Function and Molecular Composition by Tropomyosin Isoforms." Molecular Biology of the Cell 14, no. 3 (March 2003): 1002–16. http://dx.doi.org/10.1091/mbc.e02-04-0244.
Повний текст джерелаАнотація:
The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5NM1), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5NM1was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function.
2
Bridge, Dacie R., Karen H. Martin, Elizabeth R. Moore, Wendy M. Lee, James A. Carroll, Claudia L. Rocha, and Joan C. Olson. "Examining the Role of Actin-Plasma Membrane Association in Pseudomonas aeruginosa Infection and Type III Secretion Translocation in Migratory T24 Epithelial Cells." Infection and Immunity 80, no. 9 (June 11, 2012): 3049–64. http://dx.doi.org/10.1128/iai.00231-12.
Повний текст джерелаАнотація:
ABSTRACTThe opportunistic pathogenPseudomonas aeruginosatargets wounded epithelial barriers, but the cellular alteration that increases susceptibility toP. aeruginosainfection remains unclear. This study examined how cell migration contributes to the establishment ofP. aeruginosainfections using (i) highly migratory T24 epithelial cells as a cell culture model, (ii) mutations in the type III secretion (T3S) effector ExoS to manipulateP. aeruginosainfection, and (iii) high-resolution immunofluorescent microscopy to monitor ExoS translocation. ExoS includes both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities, andP. aeruginosacells expressing wild-type ExoS preferentially bound to the leading edge of T24 cells, where ExoS altered leading-edge architecture and actin anchoring in conjunction with interrupting T3S translocation. Inactivation of ExoS GAP activity allowedP. aeruginosato be internalized and secrete ExoS within T24 cells, but as with wild-type ExoS, translocation was limited in association with disruption of actin anchoring. Inactivation of ExoS ADPRT activity resulted in significantly enhanced T3S translocation byP. aeruginosacells that remained extracellular and in conjunction with maintenance of actin-plasma membrane association. Infection withP. aeruginosaexpressing ExoS lacking both GAP and ADPRT activities resulted in the highest level of T3S translocation, and this occurred in conjunction with the entry and alignment ofP. aeruginosaand ExoS along actin filaments. Collectively, in using ExoS mutants to modulate and visualize T3S translocation, we were able to (i) confirm effector secretion by internalizedP. aeruginosa, (ii) differentiate the mechanisms underlying the effects of ExoS GAP and ADPRT activities onP. aeruginosainternalization and T3S translocation, (iii) confirm that ExoS ADPRT activity targeted a cellular substrate that interrupted T3S translocation, (iv) visualize the ability ofP. aeruginosaand ExoS to align with actin filaments, and (v) demonstrate an association between actin anchoring at the leading edge of T24 cells and the establishment ofP. aeruginosainfection. Our studies also highlight the contribution of ExoS to the opportunistic nature ofP. aeruginosainfection through its ability to exert cytotoxic effects that interrupt T3S translocation andP. aeruginosainternalization, which in turn limit theP. aeruginosainfectious process.
3
Ivanov, Andrei I., Dirk Hunt, Markus Utech, Asma Nusrat, and Charles A. Parkos. "Differential Roles for Actin Polymerization and a Myosin II Motor in Assembly of the Epithelial Apical Junctional Complex." Molecular Biology of the Cell 16, no. 6 (June 2005): 2636–50. http://dx.doi.org/10.1091/mbc.e05-01-0043.
Повний текст джерелаАнотація:
Differentiation and polarization of epithelial cells depends on the formation of the apical junctional complex (AJC), which is composed of the tight junction (TJ) and the adherens junction (AJ). In this study, we investigated mechanisms of actin reorganization that drive the establishment of AJC. Using a calcium switch model, we observed that formation of the AJC in T84 intestinal epithelial cells began with the assembly of adherens-like junctions followed by the formation of TJs. Early adherens-like junctions and TJs readily incorporated exogenous G-actin and were disassembled by latrunculin B, thus indicating dependence on continuous actin polymerization. Both adherens-like junctions and TJs were enriched in actin-related protein 3 and neuronal Wiskott-Aldrich syndrome protein (N-WASP), and their assembly was prevented by the N-WASP inhibitor wiskostatin. In contrast, the formation of TJs, but not adherens-like junctions, was accompanied by recruitment of myosin II and was blocked by inhibition of myosin II with blebbistatin. In addition, blebbistatin inhibited the ability of epithelial cells to establish a columnar phenotype with proper apico-basal polarity. These findings suggest that actin polymerization directly mediates recruitment and maintenance of AJ/TJ proteins at intercellular contacts, whereas myosin II regulates cell polarization and correct positioning of the AJC within the plasma membrane.
4
Pittenger, M. F., A. Kistler, and D. M. Helfman. "Alternatively spliced exons of the beta tropomyosin gene exhibit different affinities for F-actin and effects with nonmuscle caldesmon." Journal of Cell Science 108, no. 10 (October 1, 1995): 3253–65. http://dx.doi.org/10.1242/jcs.108.10.3253.
Повний текст джерелаАнотація:
The rat beta-tropomyosin (TM) gene expresses two isoforms via alternative RNA splicing, namely skeletal muscle beta-TM and fibroblast TM-1. The latter is also expressed in smooth muscle where it corresponds to smooth muscle beta-TM. Skeletal muscle beta-TM contains exons 7 and 10, whereas exons 6 and 11 are used in fibroblasts and smooth muscle. In order to study the properties of the alternatively spliced proteins, recombinant TMs derived from bacterial and insect cell expression systems were produced, including the normal beta gene products, fibroblast TM-1 and beta skeletal muscle TM, two carboxy-terminal chimeric TMs, TM-6/10 and TM-7/11, as well as a carboxyl-truncated version of each, TM-6Cla and TM-7Cla. The purified TM isoforms were used in actin filament association studies. The apparent TM association constants (Ka) were taken as the free concentration at half saturation and were found to be 6 microM for beta Sk TM, 8.5 for TM-6/10, 25 microM for TM-1, and 30 microM for TM-7/11 at an F-actin concentration of 42 microM. For the truncated TMs, the values determined were higher still but the binding was not carried out to full saturation. Isoforms were also produced using the baculovirus-insect cell system which produces proteins with an acetylated amino terminus as is normally found in vivo. This modification significantly enhanced the F-actin association of TM-1 but not the beta skeletal TM or the other isoforms. Fibroblast TM-2 or TM-3, both products of the alpha gene, enhanced the affinity of TM-1 for F-actin, demonstrating different isoforms can act cooperatively on binding to actin. This effect was not detected with the other expressed beta gene products. The presence of 83 kDa nonmuscle caldesmon was found to enhance the binding of TM-1 for F-actin. This effect was dependent on the presence of both exons 6 and 11, as caldesmon had little effect on the other beta gene products. Collectively these results demonstrate TMs differ in their affinity for F-actin, which can be altered by other TMs or actin-binding proteins. The beta tropomyosin isoforms were fluorescently-tagged and microinjected into cultured cells to study their in vivo localization where it was found that each of the full-length TMs bound to microfilaments but, at the light microscopy level, the isoforms were not differentially localized in these fibroblasts.
5
ROTTER, Björn, Odile BOURNIER, Gael NICOLAS, Didier DHERMY та Marie-Christine LECOMTE. "αII-Spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts". Biochemical Journal 388, № 2 (24 травня 2005): 631–38. http://dx.doi.org/10.1042/bj20041502.
Повний текст джерелаАнотація:
The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of α- and β-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent α-spectrin subunit in non-erythroid cells, αII-spectrin, contains two particular spectrin repeats in its central region, α9 and α10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the α9-α10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the α10 repeat of αII-spectrin whereas EVL interacts with the Src homology 3 domain located within the α9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between αII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.
6
Bridge, Dacie R., Matthew J. Novotny, Elizabeth R. Moore, and Joan C. Olson. "Role of host cell polarity and leading edge properties in Pseudomonas type III secretion." Microbiology 156, no. 2 (February 1, 2010): 356–73. http://dx.doi.org/10.1099/mic.0.033241-0.
Повний текст джерелаАнотація:
Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen Pseudomonas aeruginosa and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchical order of protein involvement was identified that paralleled the architecture of leading edge (LE) focal complexes. Conversely, in HL-60 cells, induction of T3S sensitivity coincided with the onset of LE properties and the development of actin-rich projections associated with polarized cell migration. When LE architecture was examined by immunofluorescent staining for actin, Rac1, IQ-motif-containing GTPase-activating protein 1 (IQGAP1) and phosphatidylinositol 3 kinase (PI3 kinase), intact LE structure was found to closely correlate with host cell sensitivity to P. aeruginosa T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the LE alters membrane properties to favour T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration.
7
Jin, Sun Woo, Gi Ho Lee, Hoa Thi Pham, Jae Ho Choi, and Hye Gwang Jeong. "Polyhexamethylene Guanidine Phosphate Damages Tight Junctions and the F-Actin Architecture by Activating Calpain-1 via the P2RX7/Ca2+ Signaling Pathway." Cells 9, no. 1 (December 24, 2019): 59. http://dx.doi.org/10.3390/cells9010059.
Повний текст джерелаАнотація:
Polyhexamethylene guanidine phosphate (PHMG-p), a member of the polymeric guanidine family, has strong antimicrobial activity and may increase the risk of inflammation-associated pulmonary fibrosis. However, the effect of PHMG-p on the barrier function of the bronchial epithelium is unknown. Epithelial barrier functioning is maintained by tight junctions (TJs); damage to these TJs is the major cause of epithelial barrier breakdown during lung inflammation. The present study showed that, in BEAS-2B human bronchial epithelial cells, exposure to PHMG-p reduced the number of TJs and the E-cadherin level and impaired the integrity of the F-actin architecture. Furthermore, exposure to PHMG-p stimulated the calcium-dependent protease calpain-1, which breaks down TJs. However, treatment with the calpain-1 inhibitor, ALLN, reversed the PHMG-p-mediated impairment of TJs and the F-actin architecture. Furthermore, exposure to PHMG-p increased the intracellular Ca2+ level via P2X purinoreceptor 7 (P2RX7) and inhibition of P2RX7 abolished the PHMG-p-induced calpain-1 activity and protein degradation and increased the intracellular Ca2+ level. Although exposure to PHMG-p increased the extracellular ATP level, hydrolysis of extracellular ATP by apyrase did not influence its detrimental effect on bronchial epithelial cells. These results implicate the impairment of TJs and the F-actin architecture in the pathogenesis of pulmonary diseases.
8
Garvalov, Boyan K., Theresa E. Higgins, James D. Sutherland, Markus Zettl, Niki Scaplehorn, Thomas Köcher, Eugenia Piddini, Gareth Griffiths, and Michael Way. "The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions." Journal of Cell Biology 161, no. 1 (April 14, 2003): 33–39. http://dx.doi.org/10.1083/jcb.200211015.
Повний текст джерелаАнотація:
The function of the human Tes protein, which has extensive similarity to zyxin in both sequence and domain organization, is currently unknown. We now show that Tes is a component of focal adhesions that, when expressed, negatively regulates proliferation of T47D breast carcinoma cells. Coimmunoprecipitations demonstrate that in vivo Tes is complexed with actin, Mena, and vasodilator-stimulated phosphoprotein (VASP). Interestingly, the isolated NH2-terminal half of Tes pulls out α-actinin and paxillin from cell extracts in addition to actin. The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts. These differences suggest that the ability of Tes to associate with α-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule. Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo. Using fibroblasts lacking Mena and VASP, we show that these proteins are not required to recruit Tes to focal adhesions. However, using RNAi ablation, we demonstrate that zyxin is required to recruit Tes, as well as Mena and VASP, but not vinculin or paxillin, to focal adhesions.
9
Pittenger, M. F., and D. M. Helfman. "In vitro and in vivo characterization of four fibroblast tropomyosins produced in bacteria: TM-2, TM-3, TM-5a, and TM-5b are co-localized in interphase fibroblasts." Journal of Cell Biology 118, no. 4 (August 15, 1992): 841–58. http://dx.doi.org/10.1083/jcb.118.4.841.
Повний текст джерелаАнотація:
Most cell types express several tropomyosin isoforms, the individual functions of which are poorly understood. In rat fibroblasts there are at least six isoforms; TM-1, TM-2, TM-3, TM-4, TM-5a, and TM-5b. TM-1 is the product of the beta gene. TM-4 is produced from the TM-4 gene, and TMs 2, 3, 5a, and 5b are the products of the alpha gene. To begin to study the localization and function of the isoforms in fibroblasts, cDNAs for TM isoforms 2, 3, 5a, and 5b were placed into bacterial expression vectors and used to produce TM isoforms. The bacterially produced TMs were determined to be full length by sequencing the amino- and carboxy termini. These TMs were found to bind to F-actin in vitro, with properties similar to that of skeletal muscle TM. In addition, competition experiments demonstrated that TM-5b was better than TM-5a in displacing other TM isoforms from F-actin in vitro. To investigate the intracellular localization of these fibroblast isoforms, each was derivatized with a fluorescent chromophore and microinjected into rat fibroblasts. TM-2, TM-3, TM-5a, and TM-5b were each found to associate along actin filaments. There was no preferred cellular location or subset of actin filaments for these isoforms. Furthermore, co-injection of two isoforms labeled with different fluorochromes showed identical staining. At the level of the light microscope, these isoforms from the alpha gene do not appear to achieve different functions by binding to particular subsets of actin filaments or locations in cells. Some alternative possibilities are discussed. The results show that bacterially produced TMs can be used to study in vitro and in vivo properties of the isoforms.
10
Terai, Tomoya, Noriyuki Nishimura, Ikuno Kanda, Natsuo Yasui, and Takuya Sasaki. "JRAB/MICAL-L2 Is a Junctional Rab13-binding Protein Mediating the Endocytic Recycling of Occludin." Molecular Biology of the Cell 17, no. 5 (May 2006): 2465–75. http://dx.doi.org/10.1091/mbc.e05-09-0826.
Повний текст джерелаАнотація:
The dynamic turnover of tight junctions (TJs) is essential for epithelial-mesenchymal transitions and/or mesenchymal-epithelial transitions during epithelial morphogenesis. We previously demonstrated that Rab13 specifically mediates the endocytic recycling of occludin. Here, we identified MICAL-L2 (molecule interacting with CasL-like 2) as a novel Rab13-binding protein. Immunoprecipitation and immunofluorescence microscopy showed that MICAL-L2 specifically bound to the GTP-bound form of Rab13 via its C terminus, which contained a coiled-coil domain, and localized at TJs in epithelial MTD-1A cells. Recycling assay demonstrated that a MICAL-L2 mutant lacking the Rab13-binding domain (MICAL-L2-N) specifically inhibited the endocytic recycling of occludin but not transferrin receptor. Ca2+ switch assay further revealed that MICAL-L2-N as well as Rab13 Q67L inhibited the recruitment of occludin to the plasma membrane, the development of transepithelial electrical resistance, and the formation of a paracellular diffusion barrier. MICAL-L2 was displaced from TJs upon actin depolymerization and was distributed along radiating actin cables and stress fibers in Ca2+-depleted MTD-1A and fibroblastic NIH3T3 cells, respectively. These results suggest that MICAL-L2 mediates the endocytic recycling of occludin and the formation of functional TJs by linking Rab13 to actin cytoskeleton. We rename MICAL-L2 as JRAB (junctional Rab13-binding protein).
11
Helfman, D. M., C. Berthier, J. Grossman, M. Leu, E. Ehler, E. Perriard, and J. C. Perriard. "Nonmuscle tropomyosin-4 requires coexpression with other low molecular weight isoforms for binding to thin filaments in cardiomyocytes." Journal of Cell Science 112, no. 3 (February 1, 1999): 371–80. http://dx.doi.org/10.1242/jcs.112.3.371.
Повний текст джерелаАнотація:
Vertebrate tropomyosins (TMs) are expressed from four genes, and at least 18 distinct isoforms are generated via a complex pattern of alternative RNA splicing and alternative promoters. The functional significance of this isoform diversity is largely unknown and it remains to be determined whether specific isoforms are required for assembly and integration into distinct actin-containing structures. The ability of nonmuscle (TM-1, -2, -3, -4, -5(NM1), -5a or -5b) and striated muscle (skeletal muscle (α)-TM) isoforms to incorporate into actin filaments of neonatal rat cardiomyocytes (NRCs) was studied using expression plasmids containing TM-fusions with GFP (green fluorescent protein) as well as with VSV- or HA-epitope tags. All isoforms, except of fibroblast TM-4, were able to incorporate into the I-band of NRCs. When TM-4 was co-transfected with other low molecular weight (LMW) isoforms of TM (TM-5, TM-5a and TM-5b), it was able to incorporate into sarcomeres of NRCs. This result was not obtained when TM-4 was co-transfected with high molecular weight (HMW) TMs (TM-1, TM-2 or skeletal muscle (α)-TM). These data demonstrate that the ability of TM-4 to bind to actin filaments can be specifically influenced by its interaction with other LMW TM isoforms. In addition, cells that incorporated the muscle or nonmuscle GFP-TMs into their sarcomeres continued to beat and exhibited sarcomeric contraction. These studies provide the first in vivo demonstration of synergistic effects between TM isoforms for binding to actin filaments. These results have important implications in understanding actin filament dynamics in nonmuscle cell systems, especially during development and in transformed cells, where alterations in the ratio of different LMW isoforms might lead to changes in their interactions with actin filaments. Furthermore, these studies demonstrate that GFP-TM can be used to study thin-filament dynamics in muscle cells and actin filament dynamics in nonmuscle cells.
12
Baranwal, Somesh, Nayden G. Naydenov, Gianni Harris, Vera Dugina, Kathleen G. Morgan, Christine Chaponnier та Andrei I. Ivanov. "Nonredundant roles of cytoplasmic β- and γ-actin isoforms in regulation of epithelial apical junctions". Molecular Biology of the Cell 23, № 18 (15 вересня 2012): 3542–53. http://dx.doi.org/10.1091/mbc.e12-02-0162.
Повний текст джерелаАнотація:
Association with the actin cytoskeleton is critical for normal architecture and dynamics of epithelial tight junctions (TJs) and adherens junctions (AJs). Epithelial cells express β-cytoplasmic (β-CYA) and γ-cytoplasmic (γ-CYA) actins, which have different cellular localization and functions. This study elucidates the roles of cytoplasmic actins in regulating structure and remodeling of AJs and TJs in model intestinal epithelia. Immunofluorescence labeling and latrunculin B treatment reveal affiliation of dynamic β-CYA filaments with newly assembled and mature AJs, whereas an apical γ-CYA pool is composed of stable perijunctional bundles and rapidly turning-over nonjunctional filaments. The functional effects of cytoplasmic actins on epithelial junctions are examined by using isoform-specific small interfering RNAs and cell-permeable inhibitory peptides. These experiments demonstrate unique roles of β-CYA and γ-CYA in regulating the steady-state integrity of AJs and TJs, respectively. Furthermore, β-CYA is selectively involved in establishment of apicobasal cell polarity. Both actin isoforms are essential for normal barrier function of epithelial monolayers, rapid AJ/TJ reassembly, and formation of three-dimensional cysts. Cytoplasmic actin isoforms play unique roles in regulating structure and permeability of epithelial junctions.
13
Gokhin, David S., та Velia M. Fowler. "Cytoplasmic γ-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers". Journal of Cell Biology 194, № 1 (4 липня 2011): 105–20. http://dx.doi.org/10.1083/jcb.201011128.
Повний текст джерелаАнотація:
The sarcoplasmic reticulum (SR) serves as the Ca2+ reservoir for muscle contraction. Tropomodulins (Tmods) cap filamentous actin (F-actin) pointed ends, bind tropomyosins (Tms), and regulate F-actin organization. In this paper, we use a genetic targeting approach to examine the effect of Tmod1 deletion on the organization of cytoplasmic γ-actin (γcyto-actin) in the SR of skeletal muscle. In wild-type muscle fibers, γcyto-actin and Tmod3 defined an SR microdomain that was distinct from another Z line–flanking SR microdomain containing Tmod1 and Tmod4. The γcyto-actin/Tmod3 microdomain contained an M line complex composed of small ankyrin 1.5 (sAnk1.5), γcyto-actin, Tmod3, Tm4, and Tm5NM1. Tmod1 deletion caused Tmod3 to leave its SR compartment, leading to mislocalization and destabilization of the Tmod3–γcyto-actin–sAnk1.5 complex. This was accompanied by SR morphological defects, impaired Ca2+ release, and an age-dependent increase in sarcomere misalignment. Thus, Tmod3 regulates SR-associated γcyto-actin architecture, mechanically stabilizes the SR via a novel cytoskeletal linkage to sAnk1.5, and maintains the alignment of adjacent myofibrils.
14
Naydenov, Nayden G., and Andrei I. Ivanov. "Adducins Regulate Remodeling of Apical Junctions in Human Epithelial Cells." Molecular Biology of the Cell 21, no. 20 (October 15, 2010): 3506–17. http://dx.doi.org/10.1091/mbc.e10-03-0259.
Повний текст джерелаАнотація:
Epithelial adherens junctions (AJs) and tight junctions (TJs) are dynamic structures that readily undergo disintegration and reassembly. Remodeling of the AJs and TJs depends on the orchestrated dynamics of the plasma membrane with its underlying F-actin cytoskeleton, and the membrane–cytoskeleton interface may play a key role in junctional regulation. Spectrin–adducin–ankyrin complexes link membranes to the actin cytoskeleton where adducins mediate specrtrin–actin interactions. This study elucidates roles of adducins in the remodeling of epithelial junctions in human SK-CO15 colonic and HPAF-II pancreatic epithelial cell monolayers. These cells expressed the α and γ isoforms of adducin that positively regulated each others protein level and colocalized with E-cadherin and β-catenin at mature, internalized and newly assembled AJs. Small interfering RNA-mediated down-regulation of α- or γ-adducin expression significantly attenuated calcium-dependent AJ and TJ assembly and accelerated junctional disassembly triggered by activation of protein kinase C. Two mechanisms were found to mediate the impaired AJ and TJ assembly in adducin-depleted cells. One mechanism involved diminished expression and junctional recruitment of βII-spectrin, and the other mechanism involved the decrease in the amount of cellular F-actin and impaired assembly of perijunctional actin bundles. These findings suggest novel roles for adducins in stabilization of epithelial junctions and regulation of junctional remodeling.
15
Nakatsuji, Hiroyoshi, Noriyuki Nishimura, Rie Yamamura, Hiro-omi Kanayama, and Takuya Sasaki. "Involvement of Actinin-4 in the Recruitment of JRAB/MICAL-L2 to Cell-Cell Junctions and the Formation of Functional Tight Junctions." Molecular and Cellular Biology 28, no. 10 (March 10, 2008): 3324–35. http://dx.doi.org/10.1128/mcb.00144-08.
Повний текст джерелаАнотація:
ABSTRACT Tight junctions (TJs) are cell-cell adhesive structures that undergo continuous remodeling. We previously demonstrated that Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) localized at TJs and mediated the endocytic recycling of the integral TJ protein occludin and the formation of functional TJs. Here, we investigated how JRAB/MICAL-L2 was targeted to TJs. Using a series of deletion mutants, we found the plasma membrane (PM)-targeting domain within JRAB/MICAL-L2. We then identified actinin-4, which was originally isolated as an actin-binding protein associated with cell motility and cancer invasion/metastasis, as a binding protein for the PM-targeting domain of JRAB/MICAL-L2, using a yeast two-hybrid system. Actinin-4 was colocalized with JRAB/MICAL-L2 at cell-cell junctions and linked JRAB/MICAL-L2 to F-actin. Although actinin-4 bound to JRAB/MICAL-L2 without Rab13, the actinin-4-JRAB/MICAL-L2 interaction was enhanced by Rab13 activation. Depletion of actinin-4 by using small interfering RNA inhibited the recruitment of occludin to TJs during the Ca2+ switch. During the epithelial polarization after replating, JRAB/MICAL-L2 was recruited from the cytosol to cell-cell junctions. This JRAB/MICAL-L2 recruitment as well as the formation of functional TJs was delayed in actinin-4-depleted cells. These results indicate that actinin-4 is involved in recruiting JRAB/MICAL-L2 to cell-cell junctions and forming functional TJs.
16
Lechuga, Susana, Somesh Baranwal, and Andrei I. Ivanov. "Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions." American Journal of Physiology-Gastrointestinal and Liver Physiology 308, no. 9 (May 1, 2015): G745—G756. http://dx.doi.org/10.1152/ajpgi.00446.2014.
Повний текст джерелаАнотація:
Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis.
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Fujii, R. "TLS facilitates transport of mRNA encoding an actin-stabilizing protein to dendritic spines." Journal of Cell Science 118, no. 24 (December 15, 2005): 5755–65. http://dx.doi.org/10.1242/jcs.02692.
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Moyer, Jeannette D., Roberta B. Nowak, Nancy E. Kim, Sandra K. Larkin, Luanne L. Peters, John Hartwig, Frans A. Kuypers, and Velia M. Fowler. "Tropomodulin 1-null mice have a mild spherocytic elliptocytosis with appearance of Tropomodulin 3 in red blood cells and disruption of the membrane skeleton." Blood 116, no. 14 (October 7, 2010): 2590–99. http://dx.doi.org/10.1182/blood-2010-02-268458.
Повний текст джерелаАнотація:
Abstract The short actin filaments in the red blood cell (RBC) membrane skeleton are capped at their pointed ends by tropomodulin 1 (Tmod1) and coated with tropomyosin (TM) along their length. Tmod1-TM control of actin filament length is hypothesized to regulate spectrin-actin lattice organization and membrane stability. We used a Tmod1 knockout mouse to investigate the in vivo role of Tmod1 in the RBC membrane skeleton. Western blots of Tmod1-null RBCs confirm the absence of Tmod1 and show the presence of Tmod3, which is normally not present in RBCs. Tmod3 is present at only one-fifth levels of Tmod1 present on wild-type membranes, but levels of actin, TMs, adducins, and other membrane skeleton proteins remain unchanged. Electron microscopy shows that actin filament lengths are more variable with spectrin-actin lattices displaying abnormally large and more variable pore sizes. Tmod1-null mice display a mild anemia with features resembling hereditary spherocytic elliptocytosis, including decreased RBC mean corpuscular volume, cellular dehydration, increased osmotic fragility, reduced deformability, and heterogeneity in osmotic ektacytometry. Insufficient capping of actin filaments by Tmod3 may allow greater actin dynamics at pointed ends, resulting in filament length redistribution, leading to irregular and attenuated spectrin-actin lattice connectivity, and concomitant RBC membrane instability.
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Vlahovich, Nicole, Anthony J. Kee, Chris Van der Poel, Emma Kettle, Delia Hernandez-Deviez, Christine Lucas, Gordon S. Lynch, Robert G. Parton, Peter W. Gunning, and Edna C. Hardeman. "Cytoskeletal Tropomyosin Tm5NM1 Is Required for Normal Excitation–Contraction Coupling in Skeletal Muscle." Molecular Biology of the Cell 20, no. 1 (January 2009): 400–409. http://dx.doi.org/10.1091/mbc.e08-06-0616.
Повний текст джерелаАнотація:
The functional diversity of the actin microfilaments relies in part on the actin binding protein tropomyosin (Tm). The muscle-specific Tms regulate actin-myosin interactions and hence contraction. However, there is less known about the roles of the numerous cytoskeletal isoforms. We have shown previously that a cytoskeletal Tm, Tm5NM1, defines a Z-line adjacent cytoskeleton in skeletal muscle. Recently, we identified a second cytoskeletal Tm in this region, Tm4. Here we show that Tm4 and Tm5NM1 define separate actin filaments; the former associated with the terminal sarcoplasmic reticulum (SR) and other tubulovesicular structures. In skeletal muscles of Tm5NM1 knockout (KO) mice, Tm4 localization was unchanged, demonstrating the specificity of the membrane association. Tm5NM1 KO muscles exhibit potentiation of T-system depolarization and decreased force rundown with repeated T-tubule depolarizations consistent with altered T-tubule function. These results indicate that a Tm5NM1-defined actin cytoskeleton is required for the normal excitation–contraction coupling in skeletal muscle.
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Ohnishi, Hiroe, Takuo Nakahara, Kyoko Furuse, Hiroyuki Sasaki, Shoichiro Tsukita, and Mikio Furuse. "JACOP, a Novel Plaque Protein Localizing at the Apical Junctional Complex with Sequence Similarity to Cingulin." Journal of Biological Chemistry 279, no. 44 (July 30, 2004): 46014–22. http://dx.doi.org/10.1074/jbc.m402616200.
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The apical junctional complex is composed of various cell adhesion molecules and cytoplasmic plaque proteins. Using a monoclonal antibody that recognizes a chicken 155-kDa cytoplasmic antigen (p155) localizing at the apical junctional complex, we have cloned a cDNA of its mouse homologue. The full-length cDNA of mouse p155 encoded a 148-kDa polypeptide containing a coiled-coil domain with sequence similarity to cingulin, a tight junction (TJ)-associated plaque protein. We designated this protein JACOP (junction-associatedcoiled-coilprotein). Immunofluorescence staining showed that JACOP was concentrated in the junctional complex in various types of epithelial and endothelial cells. Furthermore, in the liver and kidney, JACOP was also distributed along non-junctional actin filaments. Upon immunoelectron microscopy, JACOP was found to be localized to the undercoat of TJs in the liver, but in some tissues, its distribution was not restricted to TJs but extended to the area of adherens junctions. Overexpression studies have revealed that JACOP was recruited to the junctional complex in epithelial cells and to cell-cell contacts and stress fibers in fibroblasts. These findings suggest that JACOP is involved in anchoring the apical junctional complex, especially TJs, to actin-based cytoskeletons.
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Boëda, Batiste, Phillip P. Knowles, David C. Briggs, Judith Murray-Rust, Erika Soriano, Boyan K. Garvalov, Neil Q. McDonald, and Michael Way. "Molecular Recognition of the Tes LIM2–3 Domains by the Actin-related Protein Arp7A." Journal of Biological Chemistry 286, no. 13 (January 29, 2011): 11543–54. http://dx.doi.org/10.1074/jbc.m110.171264.
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Wang, Bai-Yan, Yuan-Fang Liu, Jing-Yan Tang, Zhao-Hui Gu, Wei-Na Zhang, Zi-Guan Zhang, Qiang Wang, et al. "Genome-Wide Abnormality Patterns of B-Lineage Acute Lymphoblastic Leukemia in Adults in Comparison with Pediatric Cases." Blood 124, no. 21 (December 6, 2014): 3786. http://dx.doi.org/10.1182/blood.v124.21.3786.3786.
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Abstract BACKGROUND B-lineage acute lymphoblastic leukemia (B-ALL) represents the most common subtype in ALL. Genomic sequence information has been lacking in adult B-ALL, whose outcome remains dismal, while a comparison between genomic abnormalities in adult and pediatric groups is useful to further decipher disease mechanisms. METHODS We used whole exome sequencing (WES), copy number variation and molecular cytogenetics to catalog somatic mutations in 95 B-ALL patients (43 adults and 52 children).WES was conducted with appropriate depths for paired genomic DNA from bone marrow mononuclear cells at diagnosis and their matched peripheral blood samples during complete remission (CR) or saliva samples. Targeted deep sequencing (TDS) was performed in a validation cohort of 179 adult and 199 pediatric B-ALLs. RESULTS Eighty-four recurrent gene mutations were revealed by WES. Integrative analysis identified the involvement of 9 functional categories of genes: key fusions (KFs), epigenetic modifiers (EMs), signaling molecules (SMs), transcription factors (TFs), tumor suppressors (TSs), actin binding/cytoskeletons (ABCs), ion binding proteins (IBPs), trans-membrane proteins (TPs) and the others. Mutually cooperative or exclusive relationships were revealed among some of these categories. Genomic landscapes suggested two distinct mechanisms involved in the leukemogenesis: one is mainly driven by KFs together with mutations of TFs and TSs; the other results from the abnormalities of TFs and TSs, in cooperation with mutations of EMs, SMs, ABCs and IBPs recapitulating the role of KFs. A panel of histone/DNA methylation modifiers (HMMs) were revealed to bear potential value of relatively favorable prognosis in both adult and childhood patients. CONCLUSIONS We described the genome-wide abnormality patterns in adult B-ALL patients in comparison with those of pediatric cases, contributing to the understanding of leukemogenesis and the identification of potential new prognostic markers. Disclosures No relevant conflicts of interest to declare.
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Krigers, A., M. Demetz, P. Moser, J. Kerschbaumer, K. R. Brawanski, C. Thomé, and C. F. Freyschlag. "P12.05.B Impact of GAP-43 and actin expression on the outcome and overall survival in diffuse and anaplastic gliomas." Neuro-Oncology 24, Supplement_2 (September 1, 2022): ii77—ii78. http://dx.doi.org/10.1093/neuonc/noac174.270.
Повний текст джерелаАнотація:
Abstract Background Distant intercellular communication in gliomas is based on the expansion of tumor microtubuli (TMs), where actin forms cytoskeleton and GAP-43 mediates the axonal conus growth. We aimed to investigate the impact of GAP-43 and actin expression on overall survival (OS) as well as crucial epidemiologic, radiological and neuropathological prognostic factors. Material and Methods FFPE tissue of adult patients with diffuse and anaplastic gliomas, who underwent first surgery in our center between 2010 and 2019, were selected. GAP-43 and actin expression was analyzed using immunohistochemistry and semi-quantitatively ranked. Clinical, neuropathological as well as follow-up-data were gained from the institutional neuro-oncological database. Results 118 patients with a median age of 46 years (IqR: 35 - 57) were evaluated. 48 (41%) presented with a diffuse glioma and 70 (59%) revealed anaplasia. 96 (82%) cases presented with intermediate or strong GAP-43 expression and 78 (67%) with no or light actin expression. Tumors with higher expression of GAP-43 (p=0.024, HR=1.71/rank) and actin (p<0.001, HR=2.28/rank) showed significantly reduced OS. IDH wildtype glioma demonstrated significantly more expression of both proteins: GAP-43 (p=0.009) and actin (p<0.001). The same was confirmed for anaplasia (GAP-43 p=0.028, actin p=0.029), higher proliferation rate (GAP-43 p=0.016, actin p=0.038), contrast-enhancement in MRI (GAP-43 p=0.023, actin p=0.037) and age (GAP-43 p=0.004, actin p<0.001). Conclusion The intercellular distant communication network in diffuse and anaplastic gliomas formed by actin and GAP-43 is associated with a negative impact on overall survival and unfavorable prognostic features.
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Krigers, Aleksandrs, Matthias Demetz, Patrizia Moser, Johannes Kerschbaumer, Konstantin Brawanski, Helga Fritsch, Claudius Thomé, and Christian Freyschlag. "PATH-03. IMPACT OF GAP-43 AND ACTIN EXPRESSION ON THE OUTCOME AND OVERALL SURVIVAL IN DIFFUSE AND ANAPLASTIC GLIOMAS." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii150. http://dx.doi.org/10.1093/neuonc/noac209.576.
Повний текст джерелаАнотація:
Abstract Background. Distant intercellular communication in gliomas is based on the expansion of tumor microtubuli (TMs), where actin forms cytoskeleton and GAP-43 mediates the axonal conus growth. We aimed to investigate the impact of GAP-43 and actin expression on overall survival (OS) as well as crucial epidemiologic, radiological and neuropathological prognostic factors. Methods. FFPE tissue of adult patients with diffuse and anaplastic gliomas, who underwent first surgery in our center between 2010 and 2019, were selected. GAP-43 and actin expression was analyzed using immunohistochemistry and semi-quantitatively ranked. Clinical, neuropathological as well as follow-up-data were gained from the institutional neuro-oncological database. Results. 118 patients with a median age of 46 years (IqR: 35 – 57) were evaluated. 48 (41%) presented with a diffuse glioma and 70 (59%) revealed anaplasia. 96 (82%) cases presented with intermediate or strong GAP-43 expression and 78 (67%) with no or light actin expression. Tumors with higher expression of GAP-43 (p=0.024, HR=1.71/rank) and actin (p< 0.001, HR=2.28/rank) showed significantly reduced OS. IDH1 wildtype glioma demonstrated significantly more expression of both proteins: GAP-43 (p=0.009) and actin (p< 0.001). The same was confirmed for anaplasia (GAP-43 p=0.028, actin p=0.029), higher proliferation rate (GAP-43 p=0.016, actin p=0.038), contrast-enhancement in MRI (GAP-43 p=0.023, actin p=0.037) and age (GAP-43 p=0.004, actin p<0.001). Conclusions. The intercellular distant communication network in diffuse and anaplastic gliomas formed by actin and GAP-43 is associated with a negative impact on overall survival and with oncologically unfavorable prognostic features.
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Ngendahayo Mukiza, Clément, and J. Daniel Dubreuil. "Escherichia coli Heat-Stable Toxin b Impairs Intestinal Epithelial Barrier Function by Altering Tight Junction Proteins." Infection and Immunity 81, no. 8 (May 28, 2013): 2819–27. http://dx.doi.org/10.1128/iai.00455-13.
Повний текст джерелаАнотація:
ABSTRACTEscherichia coliheat-stable toxin b (STb) causes diarrhea in animals. STb binds to sulfatide, its receptor, and is then internalized. In the cytoplasm, through a cascade of events, STb triggers the opening of ion channels, allowing ion secretion and water loss and leading to diarrhea. Tight junctions (TJs) are well known for controlling paracellular traffic of ions and water by forming a physical intercellular barrier in epithelial cells, and some bacterial toxins are known to affect adversely TJs. The present study aimed at determining the effect of STb on TJs. T84 cells were treated for 24 h with purified STb and a nontoxic STb mutant (D30V). Transepithelial resistance (TER), paracellular flux marker, and confocal microscopy were used to analyze the effect of STb on TJs. Purified STb caused a significant reduction of TER parallel to an increase in paracellular permeability compared to the results seen in untreated cells or mutant D30V. The increased paracellular permeability was associated with a marked alteration of F-actin stress fibers. F-actin filament dissolution and condensation were accompanied by redistribution and/or fragmentation of ZO-1, claudin-1, and occludin. These changes were also observed following treatment of T84 cells with an 8-amino-acid peptide found in the STb sequence corresponding to a consensus sequence ofVibrio choleraeZot toxin. These effects were not observed with a scrambled peptide or mutant D30V. Our findings indicate that STb induces epithelial barrier dysfunction through changes in TJ proteins that could contribute to diarrhea.
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Gimona, M., Z. Lando, Y. Dolginov, J. Vandekerckhove, R. Kobayashi, A. Sobieszek, and D. M. Helfman. "Ca2+-dependent interaction of S100A2 with muscle and nonmuscle tropomyosins." Journal of Cell Science 110, no. 5 (March 1, 1997): 611–21. http://dx.doi.org/10.1242/jcs.110.5.611.
Повний текст джерелаАнотація:
Zero-length chemical crosslinking with 1-ethyl-3-[3-(dimethyl amino)propyl]carbodiimide (EDC) indicated an association of the Ca2+-binding protein S100A2 with tropomyosin (TM) in vitro. The mobility of the crosslinked product on SDS-PAGE gels indicated the formation of a 1:1 complex between S100A2 and TM and the interaction was Ca2+ dependent. Monoclonal antibodies were raised against S100A2 and used to determine its cellular localization in the porcine epithelial cell line LLC PK1. It was found that the localization of S100A2 depended on the differentiation state of the cells, being absent from actin stress fibers in sparsely seeded cultures, but present in the actin-containing microvilli characteristic of differentiated cells. Immunoprecipitations of [35S]methionine-labeled extracts using S100A2 as well as TM-specific antibodies failed to co-precipitate TM and S100A2, indicating a transient association between these two molecules in solution. Affinity chromatography of cell extracts on immobilized recombinant TMs, however, confirmed the Ca2+-dependent interaction between S100A2 and both muscle TMs as well as with high and low molecular mass nonmuscle TMs, suggesting that the binding site resides in one of the conserved regions of TM. Our data demonstrate the possible interaction of S100A2 with TM that is not bound to the microfilaments and indicate a differentiation-related function for S100A2 in LLC PK1 cells. The possible functional implications of this interaction are discussed.
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Kuga, Daisuke, Kaori Ushida, Shinji Mii, Atsushi Enomoto, Naoya Asai, Masato Nagino, Masahide Takahashi, and Masato Asai. "Tyrosine Phosphorylation of an Actin-Binding Protein Girdin Specifically Marks Tuft Cells in Human and Mouse Gut." Journal of Histochemistry & Cytochemistry 65, no. 6 (April 4, 2017): 347–66. http://dx.doi.org/10.1369/0022155417702586.
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Summary Tuft cells (TCs) are minor components of gastrointestinal epithelia, characterized by apical tufts and spool-shaped somas. The lack of reliable TC-markers has hindered the elucidation of its role. We developed site-specific and phosphorylation-status–specific antibodies against Girdin at tyrosine-1798 (pY1798) and found pY1798 immunostaining of mouse jejunum clearly depicted epithelial cells closely resembling TCs. This study aimed to validate pY1798 as a TC-marker. Double-fluorescence staining of intestines was performed with pY1798 and known TC-markers, for example, hematopoietic-prostaglandin-D-synthase (HPGDS), or doublecortin-like kinase 1 (DCLK1). Odds ratios (ORs) were calculated from cell counts to determine whether two markers were attracting (OR<1) or repelling (OR>1). In consequence, pY1798 signals strongly attracted those of known TC-markers. ORs for HPGDS in mouse stomach, small intestine, and colon were 0 for all, and 0.08 for DCLK1 in human small intestine. pY1798-positive cells in jejunum were distinct from other minor epithelial cells, including goblet, Paneth, and neuroendocrine cells. Thus, pY1798 was validated as a TC-marker. Interestingly, apoptosis inducers significantly increased relative TC frequencies despite the absence of proliferation at baseline. In conclusion, pY1798 is a novel TC-marker. Selective tyrosine phosphorylation and possible resistance to apoptosis inducers implied the activation of certain kinase(s) in TCs, which may become a clue to elucidate the enigmatic roles of TCs.
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RAGOOWANSI, R., U. KHAN, R. A. BROWN, and D. A. MCGROUTHER. "Differences in Morphology, Cytoskeletal Architecture and Protease Production Between Zone II Tendon and Synovial Fibroblasts in vitro." Journal of Hand Surgery 28, no. 5 (October 2003): 465–70. http://dx.doi.org/10.1016/s0266-7681(03)00140-2.
Повний текст джерелаАнотація:
Fibroblast migration is an integral component of the processes resulting in the formation of restrictive adhesions in the injured tendon, especially in Zone II. Pre-requisites for cell migration are an intact cytoskeleton and an ability to biochemically degrade the extra-cellular matrix. The relative characteristics of fibroblasts from the fibro-osseus sheath (SC), the tissue surrounding the tendon in Zone II, and the endotenon (TC) with respect to morphology, cytoskeletal structure and ability to produce matrix metalloproteinases (MMPs) 2 and 9 were compared in vitro. It was found that SCs were larger in size and demonstrated greater amounts of intra-cellular alpha-smooth muscle actin (α-SMA) and intra-membranous vinculin. Filamentous actin (F-actin) fibres in SCs were more densely packed and concentrated, resulting in stress fibres. The SCs also produce greater amounts of MMP-2 and MMP-9 compared to TCs. These observations imply that SCs play an active role in adhesion formation and should be specifically targeted to inhibit or treat tendon adhesions.
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Basuroy, Shyamali, Ankur Seth, Bertha Elias, Anjaparavanda P. Naren, and Radhakrishna Rao. "MAPK interacts with occludin and mediates EGF-induced prevention of tight junction disruption by hydrogen peroxide." Biochemical Journal 393, no. 1 (December 12, 2005): 69–77. http://dx.doi.org/10.1042/bj20050959.
Повний текст джерелаАнотація:
The MAPK (mitogen-activated protein kinase) pathway is a major intracellular signalling pathway involved in EGF (epithelial growth factor) receptor-mediated cell growth and differentiation. A novel function of MAPK activity in the mechanism of EGF-mediated protection of TJs (tight junctions) from H2O2 was examined in Caco-2 cell monolayers. EGF-mediated prevention of H2O2-induced increase in paracellular permeability was associated with the prevention of H2O2-induced Tyr-phosphorylation, Thr-dephosphorylation and cellular redistribution of occludin and ZO-1 (zonula occludin-1). EGF also prevented H2O2-induced disruption of the actin cytoskeleton and the dissociation of occludin and ZO-1 from the actin-rich detergent-insoluble fractions. MEK (MAPK/ERK kinase, where ERK stands for extracellular signal related kinase) inhibitors, PD98059 and U0126, completely blocked these protective effects of EGF on TJs. EGF rapidly increased the levels of phosphorylated MEK (p-MEK) in detergent-soluble fractions and phosphorylated ERK (p-ERK) in detergent-insoluble fractions. p-ERK was colocalized and co-immunoprecipitated with occludin. GST (glutathione S-transferase) pull-down assay showed that the C-terminal tail of occludin binds to p-ERK in Caco-2 cell extracts. Pair-wise binding studies using recombinant proteins demonstrated that ERK1 directly interacts with the C-terminal tail of occludin. Therefore the present study shows that ERK interacts with the C-terminal region of occludin and mediates the prevention of H2O2-induced disruption of TJs by EGF.
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Griffith, Elen, Amanda S. Coutts, and Donald M. Black. "RNAi knockdown of the focal adhesion protein TES reveals its role in actin stress fibre organisation." Cell Motility and the Cytoskeleton 60, no. 3 (2005): 140–52. http://dx.doi.org/10.1002/cm.20052.
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Samak, G., T. Suzuki, A. Bhargava, and R. K. Rao. "c-Jun NH2-terminal kinase-2 mediates osmotic stress-induced tight junction disruption in the intestinal epithelium." American Journal of Physiology-Gastrointestinal and Liver Physiology 299, no. 3 (September 2010): G572—G584. http://dx.doi.org/10.1152/ajpgi.00265.2010.
Повний текст джерелаАнотація:
Gastrointestinal epithelium faces osmotic stress, both at physiological and pathophysiological conditions. JNK activation is an immediate cellular response to osmotic stress. We investigated the effect of osmotic stress on intestinal epithelial barrier function and delineated the role of JNK2 in osmotic stress-induced tight junction (TJ) regulation in Caco-2 cell monolayers and ileum of Jnk −/− and Jnk2 −/− mice. The role of JNK activation in osmotic stress-induced TJ disruption was evaluated using JNK-specific inhibitor and antisense oligonucleotides. Furthermore, the effect of cold restraint stress in vivo on TJ integrity was determined in rats. Osmotic stress disrupted TJs and barrier function in Caco-2 cell monolayers without affecting cell viability. Osmotic stress activated JNK1 and JNK2 and the inhibition of JNK by SP600125 attenuated osmotic stress-induced TJ disruption. TJ disruption and barrier dysfunction by osmotic stress was associated with JNK-dependent remodeling of actin cytoskeleton. Knockdown of JNK2 accelerated TJ assembly and attenuated osmotic stress-induced TJ disruption in Caco-2 cell monolayers. In mouse ileum in vitro, osmotic stress increased paracellular permeability, which was attenuated by SP600125. Osmotic stress disrupted actin cytoskeleton and TJs and increased paracellular permeability in the ileum of wild-type and JNK1 −/− mice, but not in JNK2 −/− mouse ileum. Cold restraint stress activated JNK in rat ileum and caused JNK-dependent remodeling of actin cytoskeleton and redistribution of occludin and zona occluden-1 from the intercellular junctions. These results reveal the role of JNK2 in the mechanism of osmotic stress-induced TJ disruption in the intestinal epithelium.
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Winkelman, Jonathan D., Caitlin A. Anderson, Cristian Suarez, David R. Kovar, and Margaret L. Gardel. "Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism." Proceedings of the National Academy of Sciences 117, no. 41 (September 28, 2020): 25532–42. http://dx.doi.org/10.1073/pnas.2004656117.
Повний текст джерелаАнотація:
The actin cytoskeleton assembles into diverse load-bearing networks, including stress fibers (SFs), muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl- 1, and Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force sensitivity. Zyxin rapidly localizes via its LIM domains to failing SFs in cells, known as strain sites, to initiate SF repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites (SFSS) is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We identify that the LIM domain-containing region of 18 proteins from the Zyxin, Paxillin, Tes, and Enigma proteins accumulate to SFSS. Moreover, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to SFSS in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. We then used sequence and domain analysis to demonstrate that tandem LIM domains contribute additively, for SFSS localization. Employing in vitro reconstitution, we show that the LIM domain-containing region from mammalian zyxin and fission yeast Pxl1 binds to mechanically stressed F-actin networks but does not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress.
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Hook, J., F. Lemckert, H. Qin, G. Schevzov, and P. Gunning. "Gamma Tropomyosin Gene Products Are Required for Embryonic Development." Molecular and Cellular Biology 24, no. 6 (March 15, 2004): 2318–23. http://dx.doi.org/10.1128/mcb.24.6.2318-2323.2004.
Повний текст джерелаАнотація:
ABSTRACT The actin filament system is essential for many cellular functions, including shape, motility, cytokinesis, intracellular trafficking, and tissue organization. Tropomyosins (Tms) are rod-like components of most actin filaments that differentially affect their stability and flexibility. The Tm gene family consists of four genes, αTm, βTm, γTm (Tm5 NM, where “NM” indicates “nonmuscle”), and δTm (Tm4). Multiple isoforms of the Tm family are generated by alternative splicing of three of these genes, and their expression is highly regulated. Extensive spatial and temporal sorting of Tm isoforms into different cellular compartments has been shown to occur in several cell types. We have addressed the function of the low-molecular-weight Tms encoded by the γTm gene by eliminating the corresponding amino-terminal coding sequences from this gene. Heterozygous mice were generated, and subsequent intercrossing of the F1 pups did not result in any viable homozygous knockouts. Genotype analysis of day 2.5 morulae also failed to detect any homozygous knockouts. We have failed in our attempts to delete the second allele and generate in vitro double-knockout cells, although 51 clones displayed homologous recombination back into the originally targeted locus. We therefore conclude that low-molecular-weight products from the γTm gene are essential for both embryonic development and cell survival.
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Sung, Lanping Amy, Ke-Ming Gao, Leland J. Yee, Constance J. Temm-Grove, David M. Helfman, Jim J. C. Lin, and Majid Mehrpouryan. "Tropomyosin isoform 5b is expressed in human erythrocytes: implications of tropomodulin-TM5 or tropomodulin-TM5b complexes in the protofilament and hexagonal organization of membrane skeletons." Blood 95, no. 4 (February 15, 2000): 1473–80. http://dx.doi.org/10.1182/blood.v95.4.1473.004k50_1473_1480.
Повний текст джерелаАнотація:
The human erythrocyte membrane skeleton consists of hexagonal lattices with junctional complexes containing F-actin protofilaments of approximately 33-37 nm in length. We hypothesize that complexes formed by tropomodulin, a globular capping protein at the pointed end of actin filaments, and tropomyosin (TM), a rod-like molecule of approximately 33-35 nm, may contribute to the formation of protofilaments. We have previously cloned the human tropomodulin complementary DNA and identified human TM isoform 5 (hTM5), a product of theγ-TM gene, as one of the major TM isoforms in erythrocytes. We now identify TM5b, a product of the -TM gene, to be the second major TM isoform. TM5a, the alternatively spliced isoform of the-TM gene, which differs by 1 exon and has a weaker actin-binding affinity, however, is not present. TM4, encoded by the δ-TM gene, is not present either. In sodium dodecyl sulfate–polyacrylamide gel electrophoresis, hTM5 comigrated with the slower TM major species in erythrocyte membranes, and hTM5b comigrated with the faster TM major species. TM5b, like TM5, binds strongly to tropomodulin, more so than other TM isoforms. The 2 major TM isoforms, therefore, share several common features: They have 248 residues, are approximately 33-35 nm long, and have high affinities toward F-actin and tropomodulin. These common features may be the key to the mechanism by which protofilaments are formed. Tropomodulin-TM5 or tropomodulin-TM5b complexes may stabilize F-actin in segments of approximately 33-37 nm during erythroid terminal differentiation and may, therefore, function as a molecular ruler. TM5 and TM5b further define the hexagonal geometry of the skeletal network and allow actin-regulatory functions of TMs to be modulated by tropomodulin.
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Brady, Donita C., Jamie K. Alan, James P. Madigan, Alan S. Fanning, and Adrienne D. Cox. "The Transforming Rho Family GTPase Wrch-1 Disrupts Epithelial Cell Tight Junctions and Epithelial Morphogenesis." Molecular and Cellular Biology 29, no. 4 (December 8, 2008): 1035–49. http://dx.doi.org/10.1128/mcb.00336-08.
Повний текст джерелаАнотація:
ABSTRACT Wrch-1, an atypical and transforming Rho GTPase, regulates cellular activities including proliferation and actin organization, but its functions and effectors remain poorly characterized. We show here that Wrch-1 distributes along the apical and basolateral membranes in MDCK cells and binds the cell polarity protein Par6 in a GTP-dependent manner. Activated Wrch-1 negatively regulates the kinetics of tight junction (TJ) assembly during epithelial cell polarization but has no detectable effect on overall cell polarity in confluent monolayers. It also causes a dramatic cytoskeletal reorganization and multilayering in cells grown in two-dimensional culture and disrupts cystogenesis of cells grown in three-dimensional (3D) culture. Similarly, short hairpin RNA-mediated knockdown of Wrch-1 perturbs cystogenesis in 3D culture, suggesting that tight regulation of Wrch-1 activity is necessary for normal epithelial morphogenesis. A weakly transforming effector domain mutant of activated Wrch-1 that inhibits Par6 binding abrogates the ability of Wrch-1 to disrupt TJ formation, actin organization, and epithelial morphogenesis. We hypothesize that Wrch-1-induced morphological and growth transformation may occur in part through Par6-mediated disruption of TJs and actin organization.
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Kuragano, Masahiro, Taro Q. P. Uyeda, Keiju Kamijo, Yota Murakami, and Masayuki Takahashi. "Different contributions of nonmuscle myosin IIA and IIB to the organization of stress fiber subtypes in fibroblasts." Molecular Biology of the Cell 29, no. 8 (April 15, 2018): 911–22. http://dx.doi.org/10.1091/mbc.e17-04-0215.
Повний текст джерелаАнотація:
Stress fibers (SFs) are contractile, force-generating bundled structures that can be classified into three subtypes, namely ventral SFs (vSFs), transverse arcs (TAs), and dorsal SFs. Nonmuscle myosin II (NMII) is the main component of SFs. This study examined the roles of the NMII isoforms NMIIA and NMIIB in the organization of each SF subtype in immortalized fibroblasts. Knockdown (KD) of NMIIA (a major isoform) resulted in loss of TAs from the lamella and caused the lamella to lose its flattened shape. Exogenous expression of NMIIB rescued this defect in TA formation. However, the TAs that formed on exogenous NMIIB expression in NMIIA-KD cells and the remaining TAs in NMIIB-KD cells, which mainly consisted of NMIIB and NMIIA, respectively, failed to rescue the defect in lamellar flattening. These results indicate that both isoforms are required for the proper function of TAs in lamellar flattening. KD of NMIIB resulted in loss of vSFs from the central region of the cell body, and this defect was not rescued by exogenous expression of NMIIA, indicating that NMIIA cannot replace the function of NMIIB in vSF formation. Moreover, we raised the possibility that actin filaments in vSFs are in a stretched conformation.
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Iancu, Cristian B., Mugurel C. Rusu, Laurenţiu Mogoantă , Sorin Hostiuc, and Mihai Grigoriu. "Myocardial Telocyte-Like Cells: A Review Including New Evidence." Cells Tissues Organs 206, no. 1-2 (2018): 16–25. http://dx.doi.org/10.1159/000497194.
Повний текст джерелаАнотація:
Telocytes (TCs) are a controversial cell type characterized by the presence of a particular kind of prolongations, known as telopodes, which are long, thin, and moniliform. A number of attempts has been made to establish the molecular phenotype of cardiac TCs (i.e., expression of c-kit, CD34, vimentin, PDGRFα, PDGRFβ, etc.). We designed an immunohistochemical study involving cardiac tissue samples obtained from 10 cadavers with the aim of determining whether there are TC-like interstitial cells that populate the interstitial space other than the mural microvascular cells. We applied the markers for CD31, CD34, PDGRFα, CD117/c-kit, and α-smooth muscle actin (α-SMA). We found that, in relation to two-dimensional cuts, the endothelial tubes could be misidentified as TC-like cells, the difference being the positive identification of endothelial lumina. Moreover, we found that cardiac pericytes express PDGRFα, CD117/c-kit, and α-SMA, and that they could also be misidentified as TCs when using light microscopy. We reviewed the respective values of the previously identified markers for achieving a clear-cut identification of cardiac TCs, highlighting the critical lack of specificity.
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Racca, Alice Ward, Michael J. Rynkiewicz, Nicholas LaFave, Anita Ghosh, William Lehman, and Jeffrey R. Moore. "M8R tropomyosin mutation disrupts actin binding and filament regulation: The beginning affects the middle and end." Journal of Biological Chemistry 295, no. 50 (October 5, 2020): 17128–37. http://dx.doi.org/10.1074/jbc.ra120.014713.
Повний текст джерелаАнотація:
Dilated cardiomyopathy (DCM) is associated with mutations in cardiomyocyte sarcomeric proteins, including α-tropomyosin. In conjunction with troponin, tropomyosin shifts to regulate actomyosin interactions. Tropomyosin molecules overlap via tropomyosin–tropomyosin head-to-tail associations, forming a continuous strand along the thin filament. These associations are critical for propagation of tropomyosin's reconfiguration along the thin filament and key for the cooperative switching between heart muscle contraction and relaxation. Here, we tested perturbations in tropomyosin structure, biochemistry, and function caused by the DCM-linked mutation, M8R, which is located at the overlap junction. Localized and nonlocalized structural effects of the mutation were found in tropomyosin that ultimately perturb its thin filament regulatory function. Comparison of mutant and WT α-tropomyosin was carried out using in vitro motility assays, CD, actin co-sedimentation, and molecular dynamics simulations. Regulated thin filament velocity measurements showed that the presence of M8R tropomyosin decreased calcium sensitivity and thin filament cooperativity. The co-sedimentation of actin and tropomyosin showed weakening of actin-mutant tropomyosin binding. The binding of troponin T's N terminus to the actin-mutant tropomyosin complex was also weakened. CD and molecular dynamics indicate that the M8R mutation disrupts the four-helix bundle at the head-to-tail junction, leading to weaker tropomyosin–tropomyosin binding and weaker tropomyosin–actin binding. Molecular dynamics revealed that altered end-to-end bond formation has effects extending toward the central region of the tropomyosin molecule, which alter the azimuthal position of tropomyosin, likely disrupting the mutant thin filament response to calcium. These results demonstrate that mutation-induced alterations in tropomyosin–thin filament interactions underlie the altered regulatory phenotype and ultimately the pathogenesis of DCM.
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Nunbhakdi-Craig, Viyada, Leonard Craig, Thomas Machleidt, and Estelle Sontag. "Simian Virus 40 Small Tumor Antigen Induces Deregulation of the Actin Cytoskeleton and Tight Junctions in Kidney Epithelial Cells." Journal of Virology 77, no. 5 (March 1, 2003): 2807–18. http://dx.doi.org/10.1128/jvi.77.5.2807-2818.2003.
Повний текст джерелаАнотація:
ABSTRACT There is increasing evidence that the transforming DNA tumor virus simian virus 40 (SV40) is associated with human malignancies. SV40 small tumor antigen (small t) interacts with endogenous serine/threonine protein phosphatase 2A (PP2A) and is required for the transforming activity of SV40 in epithelial cells of the lung and kidney. Here, we show that expression of SV40 small t in epithelial MDCK cells induces acute morphological changes and multilayering. Significantly, it also causes severe defects in the biogenesis and barrier properties of tight junctions (TJs) but does not prevent formation of adherens junctions. Small t-induced TJ defects are associated with a loss of PP2A from areas of cell-cell contact; altered distribution and reduced amounts of the TJ proteins ZO-1, occludin, and claudin-1; and marked disorganization of the actin cytoskeleton. Small t-mediated F-actin rearrangements encompass increased Rac-induced membrane ruffling and lamellipodia, Cdc42-initiated filopodia, and loss of Rho-dependent stress fibers. Indeed, these F-actin changes coincide with elevated levels of Rac1 and Cdc42 and decreased amounts of RhoA in small t-expressing cells. Notably, these cellular effects of small t are dependent on its interaction with endogenous PP2A. Thus, our findings provide the first evidence that, in polarized epithelial cells, expression of small t alone is sufficient to induce deregulation of Rho GTPases, F-actin, and intercellular adhesion, through interaction with endogenous PP2A. Because defects in the actin cytoskeleton and TJ disruption have been linked to loss of cell polarity and tumor invasiveness, their deregulation by PP2A and small t likely contributes to the role of SV40 in epithelial cell transformation.
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Jennings, Brett L., Larry J. Anderson, Anne M. Estes, Xiao R. Fang, Chi Young Song, William B. Campbell, and Kafait U. Malik. "Involvement of cytochrome P-450 1B1 in renal dysfunction, injury, and inflammation associated with angiotensin II-induced hypertension in rats." American Journal of Physiology-Renal Physiology 302, no. 4 (February 15, 2012): F408—F420. http://dx.doi.org/10.1152/ajprenal.00542.2011.
Повний текст джерелаАнотація:
We investigated the contribution of cytochrome P-450 1B1 (CYP1B1) to renal dysfunction and organ damage associated with ANG II-induced hypertension in rats. ANG II (300 ng·kg−1·min−1) or vehicle were infused for 2 wk, with daily injections of a selective CYP1B1 inhibitor, 2,4,3′,5′-tetramethoxystilbene (TMS; 300 μg/kg ip), or its vehicle. ANG II increased blood pressure and renal CYP1B1 activity that were prevented by TMS. ANG II also increased water intake and urine output, decreased glomerular filtration rate, increased urinary Na+ and K+ excretion, and caused proteinuria, all of which were prevented by TMS. ANG II infusion caused hypertrophy, endothelial dysfunction, and increased reactivity of renal and interlobar arteries to vasoconstrictor agents and renal vascular resistance and interstitial fibrosis as indicated by accumulation of α-smooth muscle actin, fibronectin, and collagen, and inflammation as indicated by increased infiltration of CD-3+ cells; these effects were inhibited by TMS. ANG II infusion also increased production of reactive oxygen species (ROS) and activities of NADPH oxidase, ERK1/2, p38 MAPK, and c-Src that were prevented by TMS. TMS alone had no effect on any of the above parameters. These data suggest that CYP1B1 contributes to the renal pathophysiological changes associated with ANG II-induced hypertension, most likely via increased ROS production and activation of ERK1/2, p38 MAPK, and c-Src and that CYP1B1 could serve as a novel target for treating renal disease associated with hypertension.
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Toader, Rusu, Mogoantă, Hostiuc, Jianu, and Ilie. "An Immunohistochemical Study of Gastric Mucosa and Critical Review Indicate that the Subepithelial Telocytes are Prelymphatic Endothelial Cells." Medicina 55, no. 7 (June 27, 2019): 316. http://dx.doi.org/10.3390/medicina55070316.
Повний текст джерелаАнотація:
There are only a few studies regarding gut subepithelial telocytes (TCs). The telopodes, namely peculiar TCs’ prolongations described on two-dimensional cuts, are not enough to differentiate this specific cell type. Subepithelial TCs were associated with the intestinal stem niche but a proper differential diagnosis with lymphatic endothelial cells (LECs) was not performed. In this study, we will critically review studies suggesting that distinctive TCs could be positioned within the lamina propria. Additionally, we performed an immunohistochemical study of human gastric mucosa to test the expression of D2-40, the lymphatic marker, as well as that of CD31, CD34, CD44, CD117/c-kit, α-smooth muscle actin (α-SMA) and vimentin in the gastric subepithelial niche. The results support the poorly investigated anatomy of intramural gastric lymphatics, with circumferential collectors located on both sides of the muscularis mucosae (mucosal and then submucosal) and myenteric collectors in the muscularis propria. We also found superficial epithelial prelymphatic channels bordered by D2-40+ but CD31–TC-like cells. Deep epithelial lymphatic collectors drain in collectors within the lamina propria. Blood endothelial cells expressed CD31, CD34, CD44, and vimentin. Therefore, the positive diagnosis of TC for subepithelial CD34+ cells should be regarded with caution, as they could also be artefacts, resulting from the two-dimensional examination of three dimensional structures, or as LECs. Lymphatic markers should be routinely used to discriminate TCs from LECs.
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Karaba, Sara M., Richard C. White, and Nicholas P. Cianciotto. "Stenotrophomonas maltophilia Encodes a Type II Protein Secretion System That Promotes Detrimental Effects on Lung Epithelial Cells." Infection and Immunity 81, no. 9 (June 17, 2013): 3210–19. http://dx.doi.org/10.1128/iai.00546-13.
Повний текст джерелаАнотація:
ABSTRACTThe Gram-negative bacteriumStenotrophomonas maltophiliais increasingly identified as a multidrug-resistant pathogen, being associated with pneumonia, among other infections. Despite this increasing clinical problem, the genetic and molecular basis ofS. maltophiliavirulence is quite minimally defined. We now report that strain K279a, the first clinical isolate ofS. maltophiliato be sequenced, encodes a functional type II protein secretion (T2S) system. Indeed, mutants of K279a that contain a mutation in thexpslocus exhibit a loss of at least seven secreted proteins and three proteolytic activities. Unlike culture supernatants from the parental K279a, supernatants from multiplexpsmutants also failed to induce the rounding, detachment, and death of A549 cells, a human lung epithelial cell line. Supernatants of thexpsmutants were also unable to trigger a massive rearrangement in the host cell's actin cytoskeleton that was associated with K279a secretion. In all assays, a complementedxpsFmutant behaved as the wild type did, demonstrating that Xps T2S is required for optimal protein secretion and the detrimental effects on host cells. The activities that were defined as being Xps dependent in K279a were evident among other respiratory isolates ofS. maltophilia. Utilizing a similar type of genetic analysis, we found that a second T2S system (Gsp) encoded by the K279a genome is cryptic under all of the conditions tested. Overall, this study represents the first examination of T2S inS. maltophilia, and the data obtained indicate that Xps T2S likely plays an important role inS. maltophiliapathogenesis.
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Wu, Xiaoli, Xiaoqiang Ding, Zhishan Ding, and Ping Jia. "Total Flavonoids from Leaves of Carya Cathayensis Ameliorate Renal Fibrosis via the miR-21/Smad7 Signaling Pathway." Cellular Physiology and Biochemistry 49, no. 4 (2018): 1551–63. http://dx.doi.org/10.1159/000493458.
Повний текст джерелаАнотація:
Background/Aims: Renal tubulointerstitial fibrosis is the most common pathway of progressive kidney injury, leading to end-stage renal disease. At present, no effective prophylactic treatment method is available. This study investigated the anti-fibrotic effects of total flavonoids (TFs) extracted from leaves of Carya Cathayensis in vivo and in vitro, and explored the underlying mechanisms. Methods: Anti-fibrotic effects of TFs were measured using a mouse model of unilateral ureteral obstruction (UUO) and in transforming growth factor-β1 (TGF-β1)-treated mouse tubular epithelial cells (mTECs). mRNA expression and protein levels of Collagen I, Collagen III, and α-smooth muscle actin (α-SMA) were also tested by real-time reverse transcription PCR and western blot analysis. To elucidate the underlying mechanisms, expression of miR-21 was examined in mTECs treated with TFs using miR-21 mimics transfected into mTECs before TGF-β1 and TFs treatment. Regulation of mothers against decapentaplegic homolog (Smad) signaling by miR-21 was subsequently validated via overexpression and deletion of miR-21 followed by a luciferase assay. Results: TFs treatment attenuated renal fibrosis, and inhibited expression of collagens and α-SMA in the kidneys of mice subjected to UUO. In vitro, the TFs significantly decreased expression of fibrotic markers in TGF-β1-treated mTECs. Moreover, TFs reduced miR-21 expression in a time- and dose-dependent manner in mTECs, increased expression of Smad7, and decreased phosphorylation of Smad3. Treatment with miR-21 mimics abolished the anti-fibrotic effects of the TFs on the TGF-β1-treated mTECs. In addition, genetic deletion of miR-21 upregulated expression of Smad7 and suppressed phosphorylation of Smad3, attenuating renal fibrosis in mice. Bioinformatics predictions revealed the potential binding site of miR-21 in the 3′-untranslated region of Smad7, and this was further confirmed by the luciferase assay. Conclusion: TFs ameliorate renal fibrosis via a miR-21/Smad7 signaling pathway, indicating a potential therapy for the prevention of renal fibrosis.
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Pomerleau, V., V. Reyes-Nicolas, F. Boisvert, and N. Perreault. "A25 THE “UPSIDE-DOWN OR OUTSIDE-IN”: UNDERSTANDING HOW FOXL1+ TELOCYTES GOVERN THE EPITHELIAL-MESENCHYMAL CROSSTALK IMPACTING CELL BEHAVIOR BY USING PROTEOMICS STRATEGIES." Journal of the Canadian Association of Gastroenterology 3, Supplement_1 (February 2020): 30–31. http://dx.doi.org/10.1093/jcag/gwz047.024.
Повний текст джерелаАнотація:
Abstract Background The mechanical information contained in the basement membrane (BM) is translated into intracellular signals via a process called mechanotransduction. The integrin-mediated cellular adhesions are found at the center of this outside-in mechanism connecting specific extracellular matrix (ECM) proteins with intracellular adaptors proteins that relay the signal via F-actin cytoskeleton turnover, Rho GTPase activity and eventually down to the nucleus. Dysregulation in any step of this tightly controlled process is a major contributor of disease development. Mesenchymal Foxl1+-telocytes (TCs) are known as a communication hub between stromal and epithelial cells from their proximity to the BM and from their potential roles in epithelial mechanical support and cell signaling. We have shown that BMPR1A signaling deletion in TCs (TCΔBmpr1a) induces stem cell niche defects, stromagenesis and colonic dysplasia in mouse model of GI diseases. Thus far, no study explored TCs relevance in microenvironment biomechanics and its subsequent impact in epithelial mechanotransduction. Aims Understand how TCΔBmpr1a can modulate mechanotransduction to induce early dysplastic changes in mouse colon. Methods Matrisomics was performed to determine the inventory of ECM proteins expressed solely in the GI stromal compartment following tissue deconstruction of control and TCΔBmpr1a mice colons. Collagen fibers analysis, histological and biochemical methods were used to further characterize the matrix biodynamics. Proteomics of the associated epithelial compartment was also done to expose mechanosensors and signaling cascades affecting cell behaviour. Results Matrisomics indicate modulations in fiber assembly proteins (collagens (CL), Decorin, Biglycan), ECM remodelling enzymes (LOXL1, TGM2), growth factors (LTBP1, WNT2B) and cell adhesion mediators (Periostin). This is associated with a reorganization in CL fiber alignment, cellular delocalization of TGM2 and increased unfolded CL content. TCΔBmpr1a leads to shortcomings in matrix assembly, hence variations in the ECM architecture and a novel epithelial mechanotransduction potential. Deregulations in matrix-to-cell communication were shown by proteomic analysis of the epithelial-enriched compartment of colonic dysplastic areas, with modulations of mechanosensors such as focal adhesion components (integrins, paxillin), F-actin cytoskeleton (gelsolin, RhoA) and nuclear lamina (Prelamin A/C, nesprin). Conclusions Taken together, these results suggest that TCΔBmpr1a can reprogram epithelial cells by impacting on matrix biodynamics and epithelial mechanotransduction. Modulation in this fine regulated sequence of communication from TCs to the epithelial nucleus via ECM could lead to the etiology of GI pathologies. Funding Agencies CIHR
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Littiere, Thayssa O., Gustavo H. F. Castro, Maria del Pilar R. Rodriguez, Cristina M. Bonafé, Ana F. B. Magalhães, Rafael R. Faleiros, João I. G. Vieira, Cassiane G. Santos, and Lucas L. Verardo. "Identification and Functional Annotation of Genes Related to Horses’ Performance: From GWAS to Post-GWAS." Animals 10, no. 7 (July 10, 2020): 1173. http://dx.doi.org/10.3390/ani10071173.
Повний текст джерелаАнотація:
Integration of genomic data with gene network analysis can be a relevant strategy for unraveling genetic mechanisms. It can be used to explore shared biological processes between genes, as well as highlighting transcription factors (TFs) related to phenotypes of interest. Unlike other species, gene–TF network analyses have not yet been well applied to horse traits. We aimed to (1) identify candidate genes associated with horse performance via systematic review, and (2) build biological processes and gene–TF networks from the identified genes aiming to highlight the most candidate genes for horse performance. Our systematic review considered peer-reviewed articles using 20 combinations of keywords. Nine articles were selected and placed into groups for functional analysis via gene networks. A total of 669 candidate genes were identified. From that, gene networks of biological processes from each group were constructed, highlighting processes associated with horse performance (e.g., regulation of systemic arterial blood pressure by vasopressin and regulation of actin polymerization and depolymerization). Transcription factors associated with candidate genes were also identified. Based on their biological processes and evidence from the literature, we identified the main TFs related to horse performance traits, which allowed us to construct a gene–TF network highlighting TFs and the most candidate genes for horse performance.
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Köhler, Katja, Daniel Louvard, and Ahmed Zahraoui. "Rab13 regulates PKA signaling during tight junction assembly." Journal of Cell Biology 165, no. 2 (April 19, 2004): 175–80. http://dx.doi.org/10.1083/jcb.200312118.
Повний текст джерелаАнотація:
The GTPase Rab13 regulates the assembly of functional epithelial tight junctions (TJs) through a yet unknown mechanism. Here, we show that expression of the GTP-bound form of Rab13 inhibits PKA-dependent phosphorylation and TJ recruitment of the vasodilator-stimulated phosphoprotein, an actin remodelling protein. We demonstrate that Rab13GTP directly binds to PKA and inhibits its activity. Interestingly, activation of PKA abrogates the inhibitory effect of Rab13 on the recruitment of vasodilator-stimulated phosphoprotein, ZO-1, and claudin1 to cell–cell junctions. Rab13 is, therefore, the first GTPase that controls PKA activity and provides an unexpected link between PKA signaling and the dynamics of TJ assembly.
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Monteiro, Ana C., Ronen Sumagin, Carl R. Rankin, Giovanna Leoni, Michael J. Mina, Dirk M. Reiter, Thilo Stehle, et al. "JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function." Molecular Biology of the Cell 24, no. 18 (September 15, 2013): 2849–60. http://dx.doi.org/10.1091/mbc.e13-06-0298.
Повний текст джерелаАнотація:
Intestinal barrier function is regulated by epithelial tight junctions (TJs), structures that control paracellular permeability. Junctional adhesion molecule-A (JAM-A) is a TJ-associated protein that regulates barrier; however, mechanisms linking JAM-A to epithelial permeability are poorly understood. Here we report that JAM-A associates directly with ZO-2 and indirectly with afadin, and this complex, along with PDZ-GEF1, activates the small GTPase Rap2c. Supporting a functional link, small interfering RNA–mediated down-regulation of the foregoing regulatory proteins results in enhanced permeability similar to that observed after JAM-A loss. JAM-A–deficient mice and cultured epithelial cells demonstrate enhanced paracellular permeability to large molecules, revealing a potential role of JAM-A in controlling perijunctional actin cytoskeleton in addition to its previously reported role in regulating claudin proteins and small-molecule permeability. Further experiments suggest that JAM-A does not regulate actin turnover but modulates activity of RhoA and phosphorylation of nonmuscle myosin, both implicated in actomyosin contraction. These results suggest that JAM-A regulates epithelial permeability via association with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and control contraction of the apical cytoskeleton.
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Luo, Gao, Ding, Liu, Du, Hou, Zhu, and Lou. "Transcriptome Sequencing Reveals the Traits of Spermatogenesis and Testicular Development in Large Yellow Croaker (Larimichthys crocea)." Genes 10, no. 12 (November 21, 2019): 958. http://dx.doi.org/10.3390/genes10120958.
Повний текст джерелаАнотація:
Larimichthys crocea is an economically important marine fish in China. To date, the molecular mechanisms underlying testicular development and spermatogenesis in L. crocea have not been thoroughly elucidated. In this study, we conducted a comparative transcriptome analysis between testes (TES) and pooled multiple tissues (PMT) (liver, spleen, heart, and kidney) from six male individuals. More than 54 million clean reads were yielded from TES and PMT libraries. After mapping to the draft genome of L. crocea, we acquired 25,787 genes from the transcriptome dataset. Expression analyses identified a total of 3853 differentially expressed genes (DEGs), including 2194 testes-biased genes (highly expressed in the TES) and 1659 somatic-biased genes (highly expressed in the PMT). The dataset was further annotated by blasting with multi-databases. Functional genes and enrichment pathways involved in spermatogenesis and testicular development were analyzed, such as the neuroactive ligand–receptor interaction pathway, gonadotropin-releasing hormone (GnRH) and mitogen-activated protein kinase (MAPK) signaling pathways, cell cycle pathway, and dynein, kinesin, myosin, actin, heat shock protein (hsp), synaptonemal complex protein 2 (sycp2), doublesex- and mab-3-related transcription factor 1 (dmrt1), spermatogenesis-associated genes (spata), DEAD-Box Helicases (ddx), tudor domain-containing protein (tdrd), and piwi genes. The candidate genes identified by this study lay the foundation for further studies into the molecular mechanisms underlying testicular development and spermatogenesis in L. crocea.
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Burtnick, Leslie D., and Anita Racic. "7-Diethylamino-3-((4′-iodoacetylamino)phenyl)-4-methylcoumarin, a fluorescent probe of the hydrophobic cleft in the tropomyosin coiled coil." Canadian Journal of Chemistry 66, no. 8 (August 1, 1988): 1805–8. http://dx.doi.org/10.1139/v88-291.
Повний текст джерелаАнотація:
A sulfhydryl-specific fluorescent reagent, 7-diethylamino-3-((4′-iodoacetylamino)phenyl)-4-methylcoumarin (DCIA) was used to label cysteine residues on tropomyosin (TM) from rabbit cardiac and rabbit skeletal muscles. The emission maximum at 486 nm, the high degree of fluorescence polarization, and the limited accessibility of the bound probe to quenching by iodide suggest that the probe is bound in the hydrophobic cleft between polypeptide chains of the TM coiled coil, as well as being bound covalently at a cysteine residue. The labelled TMs retain their abilities to bind F-actin and are able to interact with deoxyribonuclease I. They, however, show a reduced tendency to aggregate into filaments in low ionic strength solutions.
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Friedrich, Colin, Nicole Endlich, Wilhelm Kriz, and Karlhans Endlich. "Podocytes are sensitive to fluid shear stress in vitro." American Journal of Physiology-Renal Physiology 291, no. 4 (October 2006): F856—F865. http://dx.doi.org/10.1152/ajprenal.00196.2005.
Повний текст джерелаАнотація:
Podocytes are exposed to mechanical forces arising from glomerular capillary pressure and filtration. It has been shown that stretch affects podocyte biology in vitro and plays a significant role in the development of glomerulosclerosis in vivo. However, whether podocytes are sensitive to fluid shear stress is completely unknown. In the present study, we therefore exposed cells of a recently generated conditionally immortalized mouse podocyte cell line to defined fluid shear stress in a flow chamber, mimicking flow of the glomerular ultrafiltrate over the surface of podocytes in Bowman's space. Shear stress above 0.25 dyne/cm2 resulted in dramatic loss of podocytes but not of proximal tubular epithelial cells (LLC-PK1 cells) after 20 h. At 0.015–0.25 dyne/cm2, lamellipodia formation in podocytes was enhanced and the actin nucleation protein cortactin was redistributed to the cell margins. Shear stress further diminished stress fibers and the presence of vinculin in focal adhesions. Linear zonula occludens-1 distribution at cell-cell contacts remained unaffected at low shear stress. At 0.25 dyne/cm2, the monolayer was broken up and remaining cell-cell contacts were reinforced by F-actin and α-actinin. Because the cytoskeletal changes induced by shear stress suggested the involvement of tyrosine kinases (TKs), we tested several TK inhibitors that were all without effect on podocyte number under static conditions. At 0.25 dyne/cm2, however, the TK inhibitors genistein and AG 82 were associated with marked podocyte loss. Our data demonstrate that podocytes are highly sensitive to fluid shear stress. Shear stress induces a reorganization of the actin cytoskeleton and activates specific tyrosine kinases that are required to withstand fluid shear stress.