Zeitschriftenartikel: „Type IV filaments“

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Ching, GY, und RK Liem. „Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments“. Journal of Cell Biology 122, Nr. 6 (15.09.1993): 1323–35. http://dx.doi.org/10.1083/jcb.122.6.1323.

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We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha-internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.

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Goosens, Vivianne J., Andreas Busch, Michaella Georgiadou, Marta Castagnini, Katrina T. Forest, Gabriel Waksman und Vladimir Pelicic. „Reconstitution of a minimal machinery capable of assembling periplasmic type IV pili“. Proceedings of the National Academy of Sciences 114, Nr. 25 (06.06.2017): E4978—E4986. http://dx.doi.org/10.1073/pnas.1618539114.

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Type IV pili (Tfp), which are key virulence factors in many bacterial pathogens, define a large group of multipurpose filamentous nanomachines widespread in Bacteria and Archaea. Tfp biogenesis is a complex multistep process, which relies on macromolecular assemblies composed of 15 conserved proteins in model gram-negative species. To improve our limited understanding of the molecular mechanisms of filament assembly, we have used a synthetic biology approach to reconstitute, in a nonnative heterologous host, a minimal machinery capable of building Tfp. Here we show that eight synthetic genes are sufficient to promote filament assembly and that the corresponding proteins form a macromolecular complex at the cytoplasmic membrane, which we have purified and characterized biochemically. Our results contribute to a better mechanistic understanding of the assembly of remarkable dynamic filaments nearly ubiquitous in prokaryotes.

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Couturier, Marc Roger, und Markus Stein. „Helicobacter pylori produces unique filaments upon host contact in vitro“. Canadian Journal of Microbiology 54, Nr. 7 (Juli 2008): 537–48. http://dx.doi.org/10.1139/w08-042.

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Helicobacter pylori exists in 2 distinct morphological states, helicoid and coccoid. Both have been observed in in vitro culture and in gastric biopsies. We visualized H. pylori during AGS cell infections using immunofluorescence microscopy. Anti-H. pylori mouse serum as well as human serum from H. pylori-positive patients recognized long, thin bacterial filaments, which formed on helicoids and more frequently on coccoids. These filaments reached lengths of 59 μm and often connected bacteria. Periodate oxidation abolished antibody recognition, suggesting that carbohydrates compose a major antigenic component of the filaments. Similar to results obtained using immunofluorescence microscopy, scanning electron microscopy imaging revealed thin filamentous structures, which were absent on uninfected cells. Both coccoid conversion and filament development increased over the time course of infection with peak filament formation at 4 h. The number of visible filaments then decreased as bacteria clustered on the apical surface of AGS cells. Since the observed filaments were clearly distinct from previously described surface structures, including flagella and the cag type IV secretion system, our results demonstrate that these filaments represent a unique, previously unrecognized, organelle.

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Kraljic, Katarina, Christopher Duckworth, Rita Tojeiro, Shadab Alam, Dmitry Bizyaev, Anne-Marie Weijmans, Nicholas Fraser Boardman und Richard R. Lane. „SDSS-IV MaNGA: 3D spin alignment of spiral and S0 galaxies“. Monthly Notices of the Royal Astronomical Society 504, Nr. 3 (21.04.2021): 4626–33. http://dx.doi.org/10.1093/mnras/stab1109.

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ABSTRACT We investigate the 3D spin alignment of galaxies with respect to the large-scale filaments using the MaNGA survey. The cosmic web is reconstructed from the Sloan Digital Sky Survey using disperse and the 3D spins of MaNGA galaxies are estimated using the thin disc approximation with integral field spectroscopy kinematics. Late-type spiral galaxies are found to have their spins parallel to the closest filament’s axis. The alignment signal is found to be dominated by low-mass spirals. Spins of S0-type galaxies tend to be oriented preferentially in perpendicular direction with respect to the filament’s axis. This orthogonal orientation is found to be dominated by S0s that show a notable misalignment between their kinematic components of stellar and ionized gas velocity fields and/or by low-mass S0s with lower rotation support compared to their high-mass counterparts. Qualitatively similar results are obtained when splitting galaxies based on the degree of ordered stellar rotation, such that galaxies with high spin magnitude have their spin aligned, and those with low spin magnitude in perpendicular direction to the filaments. In the context of conditional tidal torque theory, these findings suggest that galaxies’ spins retain memory of their larger scale environment. In agreement with measurements from hydrodynamical cosmological simulations, the measured signal at low redshift is weak, yet statistically significant. The dependence of the spin-filament orientation of galaxies on their stellar mass, morphology, and kinematics highlights the importance of sample selection to detect the signal.

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Braun, Tatjana, Matthijn R. Vos, Nir Kalisman, Nicholas E. Sherman, Reinhard Rachel, Reinhard Wirth, Gunnar F. Schröder und Edward H. Egelman. „Archaeal flagellin combines a bacterial type IV pilin domain with an Ig-like domain“. Proceedings of the National Academy of Sciences 113, Nr. 37 (30.08.2016): 10352–57. http://dx.doi.org/10.1073/pnas.1607756113.

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The bacterial flagellar apparatus, which involves ∼40 different proteins, has been a model system for understanding motility and chemotaxis. The bacterial flagellar filament, largely composed of a single protein, flagellin, has been a model for understanding protein assembly. This system has no homology to the eukaryotic flagellum, in which the filament alone, composed of a microtubule-based axoneme, contains more than 400 different proteins. The archaeal flagellar system is simpler still, in some cases having ∼13 different proteins with a single flagellar filament protein. The archaeal flagellar system has no homology to the bacterial one and must have arisen by convergent evolution. However, it has been understood that the N-terminal domain of the archaeal flagellin is a homolog of the N-terminal domain of bacterial type IV pilin, showing once again how proteins can be repurposed in evolution for different functions. Using cryo-EM, we have been able to generate a nearly complete atomic model for a flagellar-like filament of the archaeon Ignicoccus hospitalis from a reconstruction at ∼4-Å resolution. We can now show that the archaeal flagellar filament contains a β-sandwich, previously seen in the FlaF protein that forms the anchor for the archaeal flagellar filament. In contrast to the bacterial flagellar filament, where the outer globular domains make no contact with each other and are not necessary for either assembly or motility, the archaeal flagellin outer domains make extensive contacts with each other that largely determine the interesting mechanical properties of these filaments, allowing these filaments to flex.

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Sheppard, Devon, Jamie-Lee Berry, Rémi Denise, Eduardo P. C. Rocha, Steve Matthews und Vladimir Pelicic. „The major subunit of widespread competence pili exhibits a novel and conserved type IV pilin fold“. Journal of Biological Chemistry 295, Nr. 19 (09.04.2020): 6594–604. http://dx.doi.org/10.1074/jbc.ra120.013316.

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Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, Streptococcus sanguinis (ComGCSS) and Streptococcus pneumoniae (ComGCSP), revealing that this pilin displays extensive structural conservation. Strikingly, ComGCSS and ComGCSP exhibit a novel type IV pilin fold that is purely helical. Results from homology modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.

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Daehnel, Katrin, Robin Harris, Lucinda Maddera und Philip Silverman. „Fluorescence assays for F-pili and their application“. Microbiology 151, Nr. 11 (01.11.2005): 3541–48. http://dx.doi.org/10.1099/mic.0.28159-0.

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Conjugative pili are extracellular filaments elaborated by Gram-negative bacteria expressing certain type IV secretion systems. They are required at the earliest stages of conjugal DNA transfer to establish specific and secure cell–cell contacts. Conjugative pili also serve as adsorption organelles for both RNA and DNA bacteriophages. Beyond these facts, the structure, formation and function of these filaments are poorly understood. This paper describes a rapid, quantitative assay for F-pili encoded by the F plasmid type IV secretion system. The assay is based on the specific lateral adsorption of icosahedral RNA bacteriophage R17 by F-pili. Bacteriophage particles conjugated with a fluorescent dye, Alexa 488, and bound to F-pili defined filaments visible by immunofluorescence microscopy. F-pili attached to F+ cells and free F-pili were both visible by this method. For quantification, cell-bound bacteriophage were separated from free bacteriophage particles by sedimentation and released by suspending cell pellets in 0·1 % SDS. Fluorescence in cell-free supernatant fractions was measured by fluorometry. The authors present a characterization of this assay and its application to F-pilus formation by cells carrying mutations in the gene for the F-pilus subunit F-pilin. Each mutation introduced a cysteine, which F-pilin normally lacks, at a different position in its primary structure. Cysteine residues in the N-terminal domain I abolished filament formation as measured by fluorescent R17 binding. This was confirmed by measurements of DNA donor activity and filamentous DNA bacteriophage infection. With one exception (G53C), cysteines elsewhere in the F-pilin primary structure did not abolish filament formation, although some mutations differentially affected F-pilus functions.

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Ching, G. Y., und R. K. Liem. „Analysis of the roles of the head domains of type IV rat neuronal intermediate filament proteins in filament assembly using domain-swapped chimeric proteins“. Journal of Cell Science 112, Nr. 13 (01.07.1999): 2233–40. http://dx.doi.org/10.1242/jcs.112.13.2233.

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Type IV neuronal intermediate filament proteins consist of alpha-internexin, which can self-assemble into filaments and the neurofilament triplet proteins, which are obligate heteropolymers, at least in rodents. These IF proteins therefore provide good systems for elucidating the mechanism of intermediate filament assembly. To analyze the roles of the head domains of these proteins in contributing to their differential assembly properties, we generated chimeric proteins by swapping the head domains between rat alpha-internexin and either rat NF-L or NF-M and examined their assembly properties in transfected cells that lack their own cytoplasmic intermediate filament network. Lalphaalpha and Malphaalpha, the chimeric proteins generated by replacing the head domain of alpha-internexin with those of NF-L and NF-M, respectively, were unable to self-assemble into filaments. In contrast, alphaLL, a chimeric NF-L protein generated by replacing the head domain of NF-L with that of alpha-internexin, was able to self-assemble into filaments, whereas MLL, a chimeric NF-L protein containing the NF-M head domain, was unable to do so. These results demonstrate that the alpha-internexin head domain is essential for alpha-internexin's ability to self-assemble. While coassembly of Lalphaalpha with NF-M and coassembly of Malphaalpha with NF-L resulted in formation of filaments, coassembly of Lalphaalpha with NF-L and coassembly of Malphaalpha with NF-M yielded punctate patterns. These coassembly results show that heteropolymeric filament formation requires that one partner has the NF-L head domain and the other partner has the NF-M head domain. Thus, the head domains of rat NF-L and NF-M play important roles in determining the obligate heteropolymeric nature of filament formation. The data obtained from these self-assembly and coassembly studies provide some new insights into the mechanism of intermediate filament assembly.

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Lamparter, Tilman, Jennifer Babian, Katrin Fröhlich, Marion Mielke, Nora Weber, Nadja Wunsch, Finn Zais et al. „The involvement of type IV pili and the phytochrome CphA in gliding motility, lateral motility and photophobotaxis of the cyanobacterium Phormidium lacuna“. PLOS ONE 17, Nr. 1 (27.01.2022): e0249509. http://dx.doi.org/10.1371/journal.pone.0249509.

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Phormidium lacuna is a naturally competent, filamentous cyanobacterium that belongs to the order Oscillatoriales. The filaments are motile on agar and other surfaces and display rapid lateral movements in liquid culture. Furthermore, they exhibit a photophobotactic response, a phototactic response towards light that is projected vertically onto the area covered by the culture. However, the molecular mechanisms underlying these phenomena are unclear. We performed the first molecular studies on the motility of an Oscillatoriales member. We generated mutants in which a kanamycin resistance cassette (KanR) was integrated in the phytochrome gene cphA and in various genes of the type IV pilin apparatus. pilM, pilN, pilQ and pilT mutants were defective in gliding motility, lateral movements and photophobotaxis, indicating that type IV pili are involved in all three kinds of motility. pilB mutants were only partially blocked in terms of their responses. pilB is the proposed ATPase for expelling of the filament in type IV pili. The genome reveals proteins sharing weak pilB homology in the ATPase region, these might explain the incomplete phenotype. The cphA mutant revealed a significantly reduced photophobotactic response towards red light. Therefore, our results imply that CphA acts as one of several photophobotaxis photoreceptors or that it could modulate the photophobotaxis response.

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Steinert, Peter M., Lyuben N. Marekov und David A. D. Parry. „Molecular Parameters of Type IV α-Internexin and Type IV-Type III α-Internexin-Vimentin Copolymer Intermediate Filaments“. Journal of Biological Chemistry 274, Nr. 3 (15.01.1999): 1657–66. http://dx.doi.org/10.1074/jbc.274.3.1657.

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Amenta, P. S., J. Gil und A. Martinez-Hernandez. „Connective tissue of rat lung. II: Ultrastructural localization of collagen types III, IV, and VI.“ Journal of Histochemistry & Cytochemistry 36, Nr. 9 (September 1988): 1167–73. http://dx.doi.org/10.1177/36.9.3403967.

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We localized collagen types III, IV, and VI in normal rat lung by light and electron immunohistochemistry. Type IV collagen was present in every basement membrane examined and was absent from all other structures. Although types III and VI had a similar distribution, being present in the interstitium of major airways, blood vessels, and alveolar septa, as in other organs, they had different morphologies. Type III collagen formed beaded fibers, 15-20 nm in diameter, whereas type VI collagen formed fine filaments, 5-10 nm in diameter. Both collagen types were found exclusively in the interstitium, often associated with thick (30-35 nm) cross-banded type I collagen fibers. Occasionally, type III fibers and type VI filaments could be found bridging from the interstitium to the adventitial aspect of some basement membranes. Furthermore, the association of collagen type VI with types I and III and basement membranes suggests that type VI may contribute to integration of the various components of the pulmonary extracellular matrix into a functional unit.

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Daum, Bertram, und Vicki Gold. „Twitch or swim: towards the understanding of prokaryotic motion based on the type IV pilus blueprint“. Biological Chemistry 399, Nr. 7 (27.06.2018): 799–808. http://dx.doi.org/10.1515/hsz-2018-0157.

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AbstractBacteria and archaea are evolutionarily distinct prokaryotes that diverged from a common ancestor billions of years ago. However, both bacteria and archaea assemble long, helical protein filaments on their surface through a machinery that is conserved at its core. In both domains of life, the filaments are required for a diverse array of important cellular processes including cell motility, adhesion, communication and biofilm formation. In this review, we highlight the recent structures of both the type IV pilus machinery and the archaellum determinedin situ. We describe the current level of functional understanding and discuss how this relates to the pressures facing bacteria and archaea throughout evolution.

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Chin, S. S., P. Macioce und R. K. Liem. „Effects of truncated neurofilament proteins on the endogenous intermediate filaments in transfected fibroblasts“. Journal of Cell Science 99, Nr. 2 (01.06.1991): 335–50. http://dx.doi.org/10.1242/jcs.99.2.335.

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The expression and assembly characteristics of carboxyl- and amino-terminal deletion mutants of rat neurofilament low Mr (NF-L) and neurofilament middle Mr (NF-M) proteins were examined by transient transfection of cultured fibroblasts. Deletion of the carboxyl-terminal tail domain of either protein indicated that this region was not absolutely essential for co-assembly into the endogenous vimentin cytoskeleton. However, deletion into the alpha-helical rod domain resulted in an inability of the mutant proteins to co-assemble with vimentin into filamentous structures. Instead, the mutant proteins appeared to be assembled into unusual tubular-vesicular structures. Additionally, these latter deletions appeared to act as dominant negative mutants which induced the collapse of the endogenous vimentin cytoskeleton as well as the constitutively expressed NF-H and NF-M cytoskeletons in stably transfected cell lines. Thus, an intact alpha-helical rod domain was essential for normal IF co-assembly whereas carboxyl-terminal deletions into this region resulted in dramatic alterations of the existing type III and IV intermediate filament cytoskeletons in vivo. Deletions from the amino-terminal end into the alpha-helical rod region gave different results. With these deletions, the transfected protein was not co-assembled into filaments and the endogenous vimentin IF network was not disrupted, indicating that these deletion mutants are recessive. The dominant negative mutants may provide a novel approach to studying intermediate filament function within living cells.

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Gimona, Mario. „An alternatively spliced exon links intermediate filaments to adhesions“. Biochemical Journal 409, Nr. 3 (15.01.2008): e1-e2. http://dx.doi.org/10.1042/bj20071674.

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Anchorage of the contractile actomyosin apparatus to the plasma membrane at discrete sites in muscle and non-muscle cells enables the transmission and conversion of force into work, such as muscle contraction and membrane deformation to regulate cell and tissue shape. Assembly, stabilization and turnover of adhesion sites are complex processes that involve structural components, a variety of signalling and adapter molecules, diverse kinases and phosphatases, and phospholipids. The dynamic turnover of adhesions also requires the frequent interaction with other filament systems of the cytoskeleton, in particular with microtubules. How the delivery and activation of all the required components is co-ordinated, however, remains to be fully understood. In the current issue of Biochemical Journal, Sun et al. provide evidence that a specific exon that is exclusively present in the α variant of the type IV intermediate filament protein synemin interacts directly with the focal adhesion protein vinculin in its active state. Interaction of adhesion components with intermediate filaments could serve as a general mechanism to regulate cell- and tissue-specific cytoskeleton-membrane attachment.

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Yurchenco, P. D., und G. C. Ruben. „Basement membrane structure in situ: evidence for lateral associations in the type IV collagen network.“ Journal of Cell Biology 105, Nr. 6 (01.12.1987): 2559–68. http://dx.doi.org/10.1083/jcb.105.6.2559.

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To determine molecular architecture of the type IV collagen network in situ, the human amniotic basement membrane has been studied en face in stereo relief by high resolution unidirectional metal shadow casting aided by antibody decoration and morphometry. The appearance of the intact basement membrane is that of a thin sheet in which there are regions of branching strands. Salt extraction further exposes these strands to reveal an extensive irregular polygonal network that can be specifically decorated with gold-conjugated anti-type IV collagen antibody. At high magnification one sees that the network, which contains integral (9-11 nm net diameter) globular domains, is formed in great part by lateral association of monomolecular filaments to form branching strands of variable but narrow diameters. Branch points are variably spaced apart by an average of 45 nm with 4.4 globular domains per micron of strand length. Monomolecular filaments (1.7-nm net diameter) often appear to twist around each other along the strand axis; we propose that super helix formation is an inherent characteristic of lateral assembly. A previous study (Yurchenco, P. D., and H. Furthmayr. 1984. Biochemistry. 23:1839) presented evidence that purified murine type IV collagen dimers polymerize to form polygonal arrays of laterally as well as end-domain-associated molecules. The architecture of this polymer is similar to the network seen in the amnion, with lateral binding a major contributor to each. Thus, to a first approximation, isolated type IV collagen can reconstitute in vitro the polymeric molecular architecture it assumes in vivo.

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Fairfield, Maria N., Stephen J. Jones, Nicolas Biais und Joseph L. Baker. „Investigating the Response of Type IV Pilins and Type IV Pilus Filaments to Applied Force using All-Atom Steered Molecular Dynamics Simulations“. Biophysical Journal 116, Nr. 3 (Februar 2019): 185a—186a. http://dx.doi.org/10.1016/j.bpj.2018.11.1029.

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Yang, Zhe, Wei Hu, Kevin Chen, Jing Wang, Renate Lux, Z. Hong Zhou und Wenyuan Shi. „Alanine 32 in PilA is important for PilA stability and type IV pili function in Myxococcus xanthus“. Microbiology 157, Nr. 7 (01.07.2011): 1920–28. http://dx.doi.org/10.1099/mic.0.049684-0.

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Type IV pili (TFP) are membrane-anchored filaments with a number of important biological functions. In the model organism Myxococcus xanthus, TFP act as molecular engines that power social (S) motility through cycles of extension and retraction. TFP filaments consist of several thousand copies of a protein called PilA or pilin. PilA contains an N-terminal α-helix essential for TFP assembly and a C-terminal globular domain important for its activity. The role of the PilA sequence and its structure–function relationship in TFP-dependent S motility remain active areas of research. In this study, we identified an M. xanthus PilA mutant carrying an alanine to valine substitution at position 32 in the α-helix, which produced structurally intact but retraction-defective TFP. Characterization of this mutant and additional single-residue variants at this position in PilA demonstrated the critical role of alanine 32 in PilA stability, TFP assembly and retraction.

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Raynaud, Claire, Devon Sheppard, Jamie-Lee Berry, Ishwori Gurung und Vladimir Pelicic. „PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion“. Proceedings of the National Academy of Sciences 118, Nr. 22 (24.05.2021): e2102092118. http://dx.doi.org/10.1073/pnas.2102092118.

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Type IV pili (T4P) are functionally versatile filamentous nanomachines, nearly ubiquitous in prokaryotes. They are predominantly polymers of one major pilin but also contain minor pilins whose functions are often poorly defined and likely to be diverse. Here, we show that the minor pilin PilB from the T4P of Streptococcus sanguinis displays an unusual bimodular three-dimensional structure with a bulky von Willebrand factor A–like (vWA) module “grafted” onto a small pilin module via a short loop. Structural modeling suggests that PilB is only compatible with a localization at the tip of T4P. By performing a detailed functional analysis, we found that 1) the vWA module contains a canonical metal ion–dependent adhesion site, preferentially binding Mg2+ and Mn2+, 2) abolishing metal binding has no impact on the structure of PilB or piliation, 3) metal binding is important for S. sanguinis T4P–mediated twitching motility and adhesion to eukaryotic cells, and 4) the vWA module shows an intrinsic binding ability to several host proteins. These findings reveal an elegant yet simple evolutionary tinkering strategy to increase T4P functional versatility by grafting a functional module onto a pilin for presentation by the filaments. This strategy appears to have been extensively used by bacteria, in which modular pilins are widespread and exhibit an astonishing variety of architectures.

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Bogin, Bryan, Maria Fairfield, Rebecca B. Goncalves, Kimberly Jarquin, Stephen Jones, Christopher A. Lovenduski, Kevin Marin et al. „Filaments Under Force: A Computational Molecular-Scale Investigation of Type IV Pili From Multiple Organisms“. Biophysical Journal 120, Nr. 3 (Februar 2021): 294a. http://dx.doi.org/10.1016/j.bpj.2020.11.1886.

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Collyn, François, Marie-Annick Léty, Shamila Nair, Vincent Escuyer, Amena Ben Younes, Michel Simonet und Michaël Marceau. „Yersinia pseudotuberculosis Harbors a Type IV Pilus Gene Cluster That Contributes to Pathogenicity“. Infection and Immunity 70, Nr. 11 (November 2002): 6196–205. http://dx.doi.org/10.1128/iai.70.11.6196-6205.2002.

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ABSTRACT Fimbriae have been shown to play an essential role in the adhesion of pathogenic gram-negative bacteria to host cells. In the enteroinvasive bacterium Yersinia pseudotuberculosis, we characterized a previously unknown 11-kb chromosomal locus involved in the synthesis of type IV pili. The locus consists of 11 open reading frames forming a polycistronic unit and encoding putative Pil proteins, PilLMNOPQRSUVW. When introduced into Escherichia coli, the Y. pseudotuberculosis operon reconstituted bundles of filaments at a pole on the bacterial surface, demonstrating that the pil locus was functional in a heterogenous genetic background. Environmental factors regulated transcription of the Y. pseudotuberculosis operon; in particular, temperature, osmolarity, and oxygen tension were critical cues. Deletion of the type IV pilus gene cluster was associated with a reduction of Y. pseudotuberculosis pathogenicity for mice infected orally. Forty-one percent of Y. pseudotuberculosis strains isolated from human or animal sources harbored the type IV pilus locus. Therefore, the pil locus of Y. pseudotuberculosis might constitute an “adaptation island,” permitting the microorganism to colonize a vast reservoir.

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Karuppiah, Vijaykumar, und Jeremy P. Derrick. „Structure of the PilM-PilN Inner Membrane Type IV Pilus Biogenesis Complex from Thermus thermophilus“. Journal of Biological Chemistry 286, Nr. 27 (19.05.2011): 24434–42. http://dx.doi.org/10.1074/jbc.m111.243535.

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Type IV pili are surface-exposed filaments, which extend from a variety of bacterial pathogens and play a major role in pathogenesis, motility, and DNA uptake. Here, we present the crystal structure of a complex between a cytoplasmic component of the type IV pilus biogenesis system from Thermus thermophilus, PilM, in complex with a peptide derived from the cytoplasmic portion of the inner membrane protein PilN. PilM also binds ATP, and its structure is most similar to the actin-like protein FtsA. PilN binds in a narrow channel between the 1A and 1C subdomains in PilM; the binding site is well conserved in other Gram-negative bacteria, notably Neisseria meningitidis, Pseudomonas aeruginosa, and Vibrio cholerae. We find no evidence for the catalysis of ATP hydrolysis by PilM; fluorescence data indicate that the protein is likely to be saturated by ATP at physiological concentrations. In addition, binding of the PilN peptide appears to influence the environment of the ATP binding site. This is the first reported structure of a complex between two type IV pilus biogenesis proteins. We propose a model in which PilM binds ATP and then PilN as one of the first steps in the formation of the inner membrane platform of the type IV pilus biogenesis complex.

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Oikarinen, A., und L. Peltonen. „Basement membrane components and keratin in the dominantly inherited form of cylindroma“. Acta Dermato-Venereologica 65, Nr. 2 (01.03.1985): 121–25. http://dx.doi.org/10.2340/0001555565121125.

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Specific antibodies against basement membrane associated, connective tissue components: type IV and V collagens, laminin, fibronectin and heparan sulphate proteoglycan were used to study the basement membrane-like structures in cylindroma lesions. All these components were immunohistochemically demonstrated as a band surrounding islands of epithelial cells and all except fibronectin also inside the islands. Antibodies to keratin filaments stained most of the cells inside the epithelial islands confirming the epithelial origin of the cells.

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Bogin, Bryan A., Christopher A. Lovenduski, Nicolas Biais und Joseph L. Baker. „Probing the Polymorphic Transition of Type IV Pilus Filaments Under Force using Coarse-Grained Molecular Dynamics Simulations“. Biophysical Journal 116, Nr. 3 (Februar 2019): 186a. http://dx.doi.org/10.1016/j.bpj.2018.11.1030.

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Yu, Xiong, Charles Goforth, Carolin Meyer, Reinhard Rachel, Reinhard Wirth, Gunnar F. Schröder und Edward H. Egelman. „Filaments from Ignicoccus hospitalis Show Diversity of Packing in Proteins Containing N-Terminal Type IV Pilin Helices“. Journal of Molecular Biology 422, Nr. 2 (September 2012): 274–81. http://dx.doi.org/10.1016/j.jmb.2012.05.031.

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Algeciras-Schimnich, Alicia, Le Shen, Bryan C. Barnhart, Andrea E. Murmann, Janis K. Burkhardt und Marcus E. Peter. „Molecular Ordering of the Initial Signaling Events of CD95“. Molecular and Cellular Biology 22, Nr. 1 (01.01.2002): 207–20. http://dx.doi.org/10.1128/mcb.22.1.207-220.2002.

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ABSTRACT Binding of either ligand or agonistic antibodies to the death receptor CD95 (APO-1/Fas) induces the formation of the death-inducing signaling complex (DISC). We now show that signal initiation of CD95 in type I cells can be further separated into at least four distinct steps. (i) The first step is ligand-induced formation of CD95 microaggregates at the cell surface. (ii) The second step is recruitment of FADD to form a DISC. This step is dependent on actin filaments. (iii) The third step involves formation of large CD95 surface clusters. This event is positively regulated by DISC-generated caspase 8. (iv) The fourth step is internalization of activated CD95 through an endosomal pathway. The latter step is again dependent on the presence of actin filaments. The data indicate that the signal initiation by CD95 is a complex process actively regulated at various levels, providing a number of new drug targets to specifically modulate CD95 signaling.

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Yi, Hong, Norman E. Williams, Virginia M. Dress und Kenneth C. Moore. „Immunolocalization of individual filament-forming proteins in the cytoskeleton of Tetrahymena“. Proceedings, annual meeting, Electron Microscopy Society of America 48, Nr. 3 (12.08.1990): 476–77. http://dx.doi.org/10.1017/s0424820100159928.

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Four polypeptides (tetrins I-IV) have been isolated from the ciliated protozoan Tetrahvmena pyriformis. These polypeptides assemble in vitro into 3-4 nm filaments identical with those present in abundance in a cytoskeletal framework associated with the feeding organelle system (oral apparatus) of this cell type. The polypeptides ranging in molecular weights from 79-89 kDa are not similar to each other in either biochemical or immunological properties. In vivo, the filaments are organized into higher order structures described as cages, cables, and networks. The specific hypothesis arises that the alternate packing arrangements may correlate with different distributions of the individual tetrin polypeptides. We report the production of monoclonal antibodies for each tetrin polypeptide, and the determination of the location of each within the cell using confocal microscopy and immunogold-silver enhancement procedures in conjunction with transmission electron microscopy (TEM).Cell samples for confocal microscopy were labelled according to conventional immunofluorescent procedures and examined with a Bio-Rad MRC-600 laser scanning confocal microscope.

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Thorner, Paul, Laurence Heidet, Fernando Moreno Merlo, Vern Edwards, Corinne Antignac und Marie-Claire Gubler. „Diffuse Leiomyomatosis of the Esophagus: Disorder of Cell-Matrix Interaction?“ Pediatric and Developmental Pathology 1, Nr. 6 (November 1998): 543–49. http://dx.doi.org/10.1007/s100249900075.

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Diffuse leiomyomatosis (DL) is rare condition characterized by proliferation of smooth muscle in the upper gastrointestinal tract. Most cases are associated with X-linked Alport syndrome and have partial deletions in the genes encoding both the α5 and α6 chains of collagen type IV. We studied aspects of cell-matrix interaction of myocytes in an esophagogastrectomy specimen from a 12-year-old patient with DL. Myocytes had central areas of cytoplasmic rarefaction, which were actin positive and desmin poor, with the reverse pattern of staining at the cell periphery. Electron microscopy (EM) showed that the areas of rarefaction consisted of disorganized aggregates of filaments. The basement membranes ranged from thickened to thinned or absent. Immunohistochemical staining for the α1–α4 chains of collagen type IV, the α1, α2, β2, and γ1 chains of laminin, nidogen, type VI collagen, and fibronectin was normal. There was loss of the α5 and α6 chains of collagen type IV and the β1 chain of laminin. Normal staining for α1, α2, α3, α4, α6, α8, and β1 integrins was noted. Staining for α5 integrin varied from normal to reduced or negative in different cells. In DL, a primary abnormality of basement membrane may be associated with disorganization of the contractile apparatus and alterations of certain integrins. This may reflect a disturbance of cell-matrix interactions that play a role in cell differentiation and internal organization.

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Del Medico, Luca, Dario Cerletti, Philipp Schächle, Matthias Christen und Beat Christen. „The type IV pilin PilA couples surface attachment and cell-cycle initiation in Caulobacter crescentus“. Proceedings of the National Academy of Sciences 117, Nr. 17 (15.04.2020): 9546–53. http://dx.doi.org/10.1073/pnas.1920143117.

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Understanding how bacteria colonize surfaces and regulate cell-cycle progression in response to cellular adhesion is of fundamental importance. Here, we use transposon sequencing in conjunction with fluorescence resonance energy transfer (FRET) microscopy to uncover the molecular mechanism for how surface sensing drives cell-cycle initiation in Caulobacter crescentus. We identify the type IV pilin protein PilA as the primary signaling input that couples surface contact to cell-cycle initiation via the second messenger cyclic di-GMP (c-di-GMP). Upon retraction of pili filaments, the monomeric pilin reservoir in the inner membrane is sensed by the 17-amino acid transmembrane helix of PilA to activate the PleC-PleD two-component signaling system, increase cellular c-di-GMP levels, and signal the onset of the cell cycle. We termed the PilA signaling sequence CIP for “cell-cycle initiating pilin” peptide. Addition of the chemically synthesized CIP peptide initiates cell-cycle progression and simultaneously inhibits surface attachment. The broad conservation of the type IV pili and their importance in pathogens for host colonization suggests that CIP peptide mimetics offer strategies to inhibit surface sensing, prevent biofilm formation and control persistent infections.

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Klimes, Anna, Ashley E. Franks, Richard H. Glaven, Hoa Tran, Christian L. Barrett, Yu Qiu, Karsten Zengler und Derek R. Lovley. „Production of pilus-like filaments in Geobacter sulfurreducens in the absence of the type IV pilin protein PilA“. FEMS Microbiology Letters 310, Nr. 1 (09.07.2010): 62–68. http://dx.doi.org/10.1111/j.1574-6968.2010.02046.x.

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Zakaria, Silsa, Rizky Stighfarrinata und Amalia Ma'rifatul Maghfiroh. „OPTIMASI PARAMETER PROSES 3D PRINTING TERHADAP KUAT TARIK FILAMENT PETG MENGGUNAKAN METODE TAGUCHI“. JUSTI (Jurnal Sistem dan Teknik Industri) 3, Nr. 4 (31.07.2023): 538. http://dx.doi.org/10.30587/justicb.v3i4.6150.

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3D Printing is the process of making products from a digital design into three-dimensional shapes that can be seen and can be held, and have volume. Where 3D Printer is a technology that has a bright future, even the technology is still growing and supporting humans for technological advancement. 3D Printer Type FDM (Fused Deposition Modelling) is very often used because it is easy to print three-dimensional objects at low cost and also filament material that has been widely available. By using the Taguchi method to find out the optimal process parameters, where finding the larger value is better in the results of the tensile test plot means and S/N ratio using analysis software and manual calculations. Using the SOVOL SV06 brand 3D Printer machine and SUNLU brand PETG filaments, it was found that the Print Speed factor or parameter greatly affects the tensile test strength according to ASTM D638 Type IV standards. Where after tensile tests and data processing both manually and with the help of software, the S/N ratio value was found with a sequence of parameters, namely Print Speed (50 mm/s), Layer Height (0.24 mm), Print Temperature (250 °C) and Infill Density (50%).

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Conradi, Fabian D., Conrad W. Mullineaux und Annegret Wilde. „The Role of the Cyanobacterial Type IV Pilus Machinery in Finding and Maintaining a Favourable Environment“. Life 10, Nr. 11 (23.10.2020): 252. http://dx.doi.org/10.3390/life10110252.

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Type IV pili (T4P) are proteinaceous filaments found on the cell surface of many prokaryotic organisms and convey twitching motility through their extension/retraction cycles, moving cells across surfaces. In cyanobacteria, twitching motility is the sole mode of motility properly characterised to date and is the means by which cells perform phototaxis, the movement towards and away from directional light sources. The wavelength and intensity of the light source determine the direction of movement and, sometimes in concert with nutrient conditions, act as signals for some cyanobacteria to form mucoid multicellular assemblages. Formation of such aggregates or flocs represents an acclimation strategy to unfavourable environmental conditions and stresses, such as harmful light conditions or predation. T4P are also involved in natural transformation by exogenous DNA, secretion processes, and in cellular adaptation and survival strategies, further cementing the role of cell surface appendages. In this way, cyanobacteria are finely tuned by external stimuli to either escape unfavourable environmental conditions via phototaxis, exchange genetic material, and to modify their surroundings to fit their needs by forming multicellular assemblies.

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Cohen-Krausz, Sara, und Shlomo Trachtenberg. „The Structure of the Archeabacterial Flagellar Filament of the Extreme Halophile Halobacterium salinarum R1M1 and Its Relation to Eubacterial Flagellar Filaments and Type IV Pili“. Journal of Molecular Biology 321, Nr. 3 (August 2002): 383–95. http://dx.doi.org/10.1016/s0022-2836(02)00616-2.

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Rossy, Tamara, Tania Distler, Lucas A. Meirelles, Joern Pezoldt, Jaemin Kim, Lorenzo Talà, Nikolaos Bouklas, Bart Deplancke und Alexandre Persat. „Pseudomonas aeruginosa type IV pili actively induce mucus contraction to form biofilms in tissue-engineered human airways“. PLOS Biology 21, Nr. 8 (01.08.2023): e3002209. http://dx.doi.org/10.1371/journal.pbio.3002209.

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The opportunistic pathogen Pseudomonas aeruginosa causes antibiotic–recalcitrant pneumonia by forming biofilms in the respiratory tract. Despite extensive in vitro experimentation, how P. aeruginosa forms biofilms at the airway mucosa is unresolved. To investigate the process of biofilm formation in realistic conditions, we developed AirGels: 3D, optically accessible tissue–engineered human lung models that emulate the airway mucosal environment. AirGels recapitulate important factors that mediate host–pathogen interactions including mucus secretion, flow and air–liquid interface (ALI), while accommodating high–resolution live microscopy. With AirGels, we investigated the contributions of mucus to P. aeruginosa biofilm biogenesis in in vivo–like conditions. We found that P. aeruginosa forms mucus–associated biofilms within hours by contracting luminal mucus early during colonization. Mucus contractions facilitate aggregation, thereby nucleating biofilms. We show that P. aeruginosa actively contracts mucus using retractile filaments called type IV pili. Our results therefore suggest that, while protecting epithelia, mucus constitutes a breeding ground for biofilms.

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Coulon, Josiane, Monique Diano, Jean-Pierre Arsanto und Yves Thouveny. „Remodeling processes during anterior regeneration of Owenia fusiformis (Polychaeta, Annelidae): a morphological and immunocytochemical survey“. Canadian Journal of Zoology 67, Nr. 4 (01.04.1989): 994–1005. http://dx.doi.org/10.1139/z89-143.

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Morphological and immunocytochemical studies were used to determine the correlations among cytoskeleton, junction complex, and basement membrane reorganization during anterior regeneration in Owenia fusiformis. Electron microscopical observations showed that, at the point of amputation, in conjunction with the disorganization of the extracellular matrix, alterations in the adhesive junctions caused dramatic changes in the distribution of intermediary filaments. Fluorescence microscopy investigations with fluorescein – phalloidin and antibodies to actin and Owenia tropomyosin visualized a basal actin network in epidermal cells, which was reorganized during regeneration. Two or three days after amputation, this cortical mat was not observed in blastema where the basal membrane was lacking. It was still incompletely reassembled after 4 or 5 days, i.e., when the thin reforming basal membrane, which stained with anti type IV collagen antibody, did not yet display collagen fibrils. In the epidermis of 6- to 7-day-old regenerates, the basal membrane, intercellular junctions, and actin and intermediary filament cytoskeletons exhibited nearly normal structural features. Use of antibody to Owenia paramyosin showed that the reorganization of the muscles during regeneration was concomitant with actin cytoskeletal reconstruction, as appeared after antitropomyosin labeling. Immunofluorescence findings were checked and (or) specified by immunogold staining.

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Llontop, Edgar E., William Cenens, Denize C. Favaro, Germán G. Sgro, Roberto K. Salinas, Cristiane R. Guzzo und Chuck S. Farah. „The PilB-PilZ-FimX regulatory complex of the Type IV pilus from Xanthomonas citri“. PLOS Pathogens 17, Nr. 8 (16.08.2021): e1009808. http://dx.doi.org/10.1371/journal.ppat.1009808.

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Type IV pili (T4P) are thin and flexible filaments found on the surface of a wide range of Gram-negative bacteria that undergo cycles of extension and retraction and participate in a variety of important functions related to lifestyle, defense and pathogenesis. During pilus extensions, the PilB ATPase energizes the polymerization of pilin monomers from the inner membrane. In Xanthomonas citri, two cytosolic proteins, PilZ and the c-di-GMP receptor FimX, are involved in the regulation of T4P biogenesis through interactions with PilB. In vivo fluorescence microscopy studies show that PilB, PilZ and FimX all colocalize to the leading poles of X. citri cells during twitching motility and that this colocalization is dependent on the presence of all three proteins. We demonstrate that full-length PilB, PilZ and FimX can interact to form a stable complex as can PilB N-terminal, PilZ and FimX C-terminal fragments. We present the crystal structures of two binary complexes: i) that of the PilB N-terminal domain, encompassing sub-domains ND0 and ND1, bound to PilZ and ii) PilZ bound to the FimX EAL domain within a larger fragment containing both GGDEF and EAL domains. Evaluation of PilZ interactions with PilB and the FimX EAL domain in these and previously published structures, in conjunction with mutagenesis studies and functional assays, allow us to propose an internally consistent model for the PilB-PilZ-FimX complex and its interactions with the PilM-PilN complex in the context of the inner membrane platform of the X. citri Type IV pilus.

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Dunger, German, Cristiane R. Guzzo, Maxuel O. Andrade, Jeffrey B. Jones und Chuck S. Farah. „Xanthomonas citri subsp. citri Type IV Pilus Is Required for Twitching Motility, Biofilm Development, and Adherence“. Molecular Plant-Microbe Interactions® 27, Nr. 10 (Oktober 2014): 1132–47. http://dx.doi.org/10.1094/mpmi-06-14-0184-r.

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Bacterial type IV pili (T4P) are long, flexible surface filaments that consist of helical polymers of mostly pilin subunits. Cycles of polymerization, attachment, and depolymerization mediate several pilus-dependent bacterial behaviors, including twitching motility, surface adhesion, pathogenicity, natural transformation, escape from immune system defense mechanisms, and biofilm formation. The Xanthomonas citri subsp. citri strain 306 genome codes for a large set of genes involved in T4P biogenesis and regulation and includes several pilin homologs. We show that X. citri subsp. citri can exhibit twitching motility in a manner similar to that observed in other bacteria such as Pseudomonas aeruginosa and Xylella fastidiosa and that this motility is abolished in Xanthomonas citri subsp. citri knockout strains in the genes coding for the major pilin subunit PilAXAC3241, the ATPases PilBXAC3239 and PilTXAC2924, and the T4P biogenesis regulators PilZXAC1133 and FimXXAC2398. Microscopy analyses were performed to compare patterns of bacterial migration in the wild-type and knockout strains and we observed that the formation of mushroom-like structures in X. citri subsp. citri biofilm requires a functional T4P. Finally, infection of X. citri subsp. citri cells by the bacteriophage (ΦXacm4-11 is T4P dependent. The results of this study improve our understanding of how T4P influence Xanthomonas motility, biofilm formation, and susceptibility to phage infection.

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Hirsch, Ann M., Rebecca SN Krupp, Yimei Lin, Susan S. Wang, Weigang Yang und Shirley C. Tucker. „Inflorescence and flower development in wild-type and sid mutant Melilotus alba, white sweetclover“. Canadian Journal of Botany 80, Nr. 7 (01.07.2002): 732–40. http://dx.doi.org/10.1139/b02-045.

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White sweetclover, Melilotus alba Desr. (Fabaceae), produces white, papilionoid flowers on a simple raceme. Individual floral apices originate in the axil of a bract. Each flower consists of five alternating whorls that, from outside to inside, consist of (i) five sepals, (ii) five petals, of which two fuse along their abaxial edges to form the keel, (iii) five antesepalous stamens, (iv) five antepetalous stamens with shorter filaments, and (v) a single carpel containing two to four ovules. The development of the wild-type sweetclover inflorescence and flowers is described in detail and compared with a mutant in which secondary inflorescences, instead of individual flowers, developed in axils of the bracts, especially at the base of the inflorescence. This white sweetclover mutant, designated sid for "secondary inflorescence development", might serve as a test of the ABC model of floral development, which was based on the model plants Antirrhinum and Arabidopsis.Key words: white sweetclover, inflorescence, flower, development, sid mutant.

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Xiao, Ke, Chuanjun Shu, Qin Yan und Xiao Sun. „Predicting Homogeneous Pilus Structure from Monomeric Data and Sparse Constraints“. BioMed Research International 2015 (2015): 1–12. http://dx.doi.org/10.1155/2015/817134.

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Type IV pili (T4P) and T2SS (Type II Secretion System) pseudopili are filaments extending beyond microbial surfaces, comprising homologous subunits called “pilins.” In this paper, we presented a new approach to predict pseudo atomic models of pili combining ambiguous symmetric constraints with sparse distance information obtained from experiments and based neither on electronic microscope (EM) maps nor on accuratea priorisymmetric details. The approach was validated by the reconstruction of the gonococcal (GC) pilus fromNeisseria gonorrhoeae, the type IVb toxin-coregulated pilus (TCP) fromVibrio cholerae, and pseudopilus of the pullulanase T2SS (the PulG pilus) fromKlebsiella oxytoca. In addition, analyses of computational errors showed that subunits should be treated cautiously, as they are slightly flexible and not strictly rigid bodies. A global sampling in a wider range was also implemented and implied that a pilus might have more than one but fewer than many possible intact conformations.

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Touhami, Ahmed, Manfred H. Jericho, Jessica M. Boyd und Terry J. Beveridge. „Nanoscale Characterization and Determination of Adhesion Forces of Pseudomonas aeruginosa Pili by Using Atomic Force Microscopy“. Journal of Bacteriology 188, Nr. 2 (15.01.2006): 370–77. http://dx.doi.org/10.1128/jb.188.2.370-377.2006.

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ABSTRACT Type IV pili play an important role in bacterial adhesion, motility, and biofilm formation. Here we present high-resolution atomic force microscopy (AFM) images of type IV pili from Pseudomonas aeruginosa bacteria. An individual pilus ranges in length from 0.5 to 7 μm and has a diameter from 4 to 6 nm, although often, pili bundles in which the individual filaments differed in both length and diameter were seen. By attaching bacteria to AFM tips, it was possible to fasten the bacteria to mica surfaces by pili tethers. Force spectra of tethered pili gave rupture forces of 95 pN. The slopes of force curves close to the rupture force were nearly linear but showed little variation with pilus length. Furthermore, force curves could not be fitted with wormlike-chain polymer stretch models when using realistic persistence lengths for pili. The observation that the slopes near rupture did not depend on the pili length suggests that they do not represent elastic properties of the pili. It is possible that this region of the force curves is determined by an elastic element that is part of the bacterial wall, although further experiments are needed to confirm this.

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Leech, Andrew J., und John S. Mattick. „Effect of Site-Specific Mutations in Different Phosphotransfer Domains of the Chemosensory Protein ChpA on Pseudomonas aeruginosa Motility“. Journal of Bacteriology 188, Nr. 24 (29.09.2006): 8479–86. http://dx.doi.org/10.1128/jb.00157-06.

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ABSTRACT The virulence of Pseudomonas aeruginosa and other surface pathogens involves the coordinate expression of a wide range of virulence determinants, including type IV pili. These surface filaments are important for the colonization of host epithelial tissues and mediate bacterial attachment to, and translocation across, surfaces by a process known as twitching motility. This process is controlled in part by a complex signal transduction system whose central component, ChpA, possesses nine potential sites of phosphorylation, including six histidine-containing phosphotransfer (HPt) domains, one serine-containing phosphotransfer domain, one threonine-containing phosphotransfer domain, and one CheY-like receiver domain. Here, using site-directed mutagenesis, we show that normal twitching motility is entirely dependent on the CheY-like receiver domain and partially dependent on two of the HPt domains. Moreover, under different assay conditions, point mutations in several of the phosphotransfer domains of ChpA give rise to unusual “swarming” phenotypes, possibly reflecting more subtle perturbations in the control of P. aeruginosa motility that are not evident from the conventional twitching stab assay. Together, these results suggest that ChpA plays a central role in the complex regulation of type IV pilus-mediated motility in P. aeruginosa.

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QIU, Feng, Anne LAKEY, Bogos AGIANIAN, Amanda HUTCHINGS, Geoffrey W. BUTCHER, Siegfried LABEIT, Kevin LEONARD und Belinda BULLARD. „Troponin C in different insect muscle types: identification of two isoforms in Lethocerus, Drosophila and Anopheles that are specific to asynchronous flight muscle in the adult insect“. Biochemical Journal 371, Nr. 3 (01.05.2003): 811–21. http://dx.doi.org/10.1042/bj20021814.

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The indirect flight muscles (IFMs) of Lethocerus (giant water bug) and Drosophila (fruitfly) are asynchronous: oscillatory contractions are produced by periodic stretches in the presence of a Ca2+ concentration that does not fully activate the muscle. The troponin complex on thin filaments regulates contraction in striated muscle. The complex in IFM has subunits that are specific to this muscle type, and stretch activation may act through troponin. Lethocerus and Drosophila have an unusual isoform of the Ca2+-binding subunit of troponin, troponin C (TnC), with a single Ca2+-binding site near the C-terminus (domain IV); this isoform is only in IFMs, together with a minor isoform with an additional Ca2+-binding site in the N-terminal region (domain II). Lethocerus has another TnC isoform in leg muscle which also has two Ca2+-binding sites. Ca2+ binds more strongly to domain IV than to domain II in two-site isoforms. There are four isoforms in Drosophila and Anopheles (malarial mosquito), three of which are also in adult Lethocerus. A larval isoform has not been identified in Lethocerus. Different TnC isoforms are expressed in the embryonic, larval, pupal and adult stages of Drosophila; the expression of the two IFM isoforms is increased in the pupal stage. Immunoelectron microscopy shows the distribution of the major IFM isoform with one Ca2+-binding site is uniform along Lethocerus thin filaments. We suggest that initial activation of IFM is by Ca2+ binding to troponin with the two-site TnC, and full activation is through the action of stretch on the complex with the one-site isoform.

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Lie, Pearl P. Y., Dolores D. Mruk, Will M. Lee und C. Yan Cheng. „Cytoskeletal dynamics and spermatogenesis“. Philosophical Transactions of the Royal Society B: Biological Sciences 365, Nr. 1546 (27.05.2010): 1581–92. http://dx.doi.org/10.1098/rstb.2009.0261.

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Different cellular events occur during spermatogenesis, and these include (i) mitosis for self-renewal of spermatogonia, (ii) differentiation of type A spermatogonia into type B and commitment of type B spermatogonia to develop into preleptotene primary spermatocytes, (iii) transit of preleptotene/leptotene spermatocytes across the blood–testis barrier in coordination with germ cell cycle progression and meiosis, (iv) spermiogenesis and spermiation. These events also associate with extensive changes in cell shape and size, and germ cell movement. The cytoskeleton, which comprises actin, microtubules and intermediate filaments, is believed to function in these cellular events. However, few studies have been conducted by investigators in the past decades to unfold the role of the cytoskeleton during spermatogenesis. This review summarizes recent advances in the field relating to cytoskeletal dynamics in the testis, and highlights areas of research that require additional emphasis so that new approaches for male contraception, as well as therapeutic approaches to alleviate environmental toxicant-induced reproductive dysfunction in men, can possibly be developed.

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Bouteiller, Mathilde, Mathias Gallique, Yvann Bourigault, Artemis Kosta, Julie Hardouin, Sebastien Massier, Yoan Konto-Ghiorghi et al. „Crosstalk between the Type VI Secretion System and the Expression of Class IV Flagellar Genes in the Pseudomonas fluorescens MFE01 Strain“. Microorganisms 8, Nr. 5 (25.04.2020): 622. http://dx.doi.org/10.3390/microorganisms8050622.

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Type VI secretion systems (T6SSs) are contractile bacterial multiprotein nanomachines that enable the injection of toxic effectors into prey cells. The Pseudomonas fluorescens MFE01 strain has T6SS antibacterial activity and can immobilise competitive bacteria through the T6SS. Hcp1 (hemolysin co-regulated protein 1), a constituent of the T6SS inner tube, is involved in such prey cell inhibition of motility. Paradoxically, disruption of the hcp1 or T6SS contractile tail tssC genes results in the loss of the mucoid and motile phenotypes in MFE01. Here, we focused on the relationship between T6SS and flagella-associated motility. Electron microscopy revealed the absence of flagellar filaments for MFE01Δhcp1 and MFE01ΔtssC mutants. Transcriptomic analysis showed a reduction in the transcription of class IV flagellar genes in these T6SS mutants. However, transcription of fliA, the gene encoding the class IV flagellar sigma factor, was unaffected. Over-expression of fliA restored the motile and mucoid phenotypes in both MFE01Δhcp1+fliA, and MFE01ΔtssC+fliA and a fliA mutant displayed the same phenotypes as MFE01Δhcp1 and MFE01ΔtssC. Moreover, the FliA anti-sigma factor FlgM was not secreted in the T6SS mutants, and flgM over-expression reduced both motility and mucoidy. This study provides arguments to unravel the crosstalk between T6SS and motility.

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Koch, Matthias D., Chenyi Fei, Ned S. Wingreen, Joshua W. Shaevitz und Zemer Gitai. „Competitive binding of independent extension and retraction motors explains the quantitative dynamics of type IV pili“. Proceedings of the National Academy of Sciences 118, Nr. 8 (16.02.2021): e2014926118. http://dx.doi.org/10.1073/pnas.2014926118.

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Type IV pili (TFP) function through cycles of extension and retraction. The coordination of these cycles remains mysterious due to a lack of quantitative measurements of multiple features of TFP dynamics. Here, we fluorescently label TFP in the pathogen Pseudomonas aeruginosa and track full extension and retraction cycles of individual filaments. Polymerization and depolymerization dynamics are stochastic; TFP are made at random times and extend, pause, and retract for random lengths of time. TFP can also pause for extended periods between two extension or two retraction events in both wild-type cells and a slowly retracting PilT mutant. We developed a biophysical model based on the stochastic binding of two dedicated extension and retraction motors to the same pilus machine that predicts the observed features of the data with no free parameters. We show that only a model in which both motors stochastically bind and unbind to the pilus machine independent of the piliation state of the machine quantitatively explains the experimentally observed pilus production rate. In experimental support of this model, we show that the abundance of the retraction motor dictates the pilus production rate and that PilT is bound to pilus machines even in their unpiliated state. Together, the strong quantitative agreement of our model with a variety of experiments suggests that the entire repetitive cycle of pilus extension and retraction is coordinated by the competition of stochastic motor binding to the pilus machine, and that the retraction motor is the major throttle for pilus production.

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Leighton, Tiffany L., Neha Dayalani, Liliana M. Sampaleanu, P. Lynne Howell und Lori L. Burrows. „Novel Role for PilNO in Type IV Pilus Retraction Revealed by Alignment Subcomplex Mutations“. Journal of Bacteriology 197, Nr. 13 (27.04.2015): 2229–38. http://dx.doi.org/10.1128/jb.00220-15.

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ABSTRACTType IV pili (T4P) are dynamic protein filaments that mediate bacterial adhesion, biofilm formation, and twitching motility. The highly conserved PilMNOP proteins form an inner membrane alignment subcomplex required for function of the T4P system, though their exact roles are unclear. Three potential interaction interfaces for PilNO were identified: core-core, coiled coils (CC), and the transmembrane segments (TMSs). A high-confidence PilNO heterodimer model was used to select key residues for mutation, and the resulting effects on protein-protein interactions were examined both in a bacterial two-hybrid (BTH) system and in their nativePseudomonas aeruginosacontext. Mutations in the oppositely charged CC regions or the TMS disrupted PilNO heterodimer formation in the BTH assay, while up to six combined mutations in the core failed to disrupt the interaction. When the mutations were introduced into theP. aeruginosachromosome at thepilNorpilOlocus, specific changes at each of the three interfaces—including core mutations that failed to disrupt interactions in the BTH system—abrogated surface piliation and/or impaired twitching motility. Unexpectedly, specific CC mutants were hyperpiliated but nonmotile, a hallmark of pilus retraction defects. These data suggest that PilNO participate in both the extension and retraction of T4P. Our findings support a model of multiple, precise interaction interfaces between PilNO; emphasize the importance of studying protein function in a minimally perturbed context and stoichiometry; and highlight potential target sites for development of small-molecule inhibitors of the T4P system.IMPORTANCEPseudomonas aeruginosais an opportunistic pathogen that uses type IV pili (T4P) for host attachment. The T4P machinery is composed of four cell envelope-spanning subcomplexes. PilN and PilO heterodimers are part of the alignment subcomplex and essential for T4P function. Three potential PilNO interaction interfaces (the core-core, coiled-coil, and transmembrane segment interfaces) were probed using site-directed mutagenesis followed by functional assays in anEscherichia colitwo-hybrid system and inP. aeruginosa. Several mutations blocked T4P assembly and/or motility, including two that revealed a novel role for PilNO in pilus retraction, while other mutations affected extension dynamics. These critical PilNO interaction interfaces represent novel targets for small-molecule inhibitors with the potential to disrupt T4P function.

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Lee, Yichen, Bo H. Lee, William Yip, Pingchen Chou und Bak-Sau Yip. „Neurofilament Proteins as Prognostic Biomarkers in Neurological Disorders“. Current Pharmaceutical Design 25, Nr. 43 (09.01.2020): 4560–69. http://dx.doi.org/10.2174/1381612825666191210154535.

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Neurofilaments: light, medium, and heavy (abbreviated as NF-L, NF-M, and NF-H, respectively), which belong to Type IV intermediate filament family (IF), are neuron-specific cytoskeletal components. Neurofilaments are axonal structural components and integral components of synapses, which are important for neuronal electric signal transmissions along the axons and post-translational modification. Abnormal assembly of neurofilaments is found in several human neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy (SMA), and hereditary sensory-motor neuropathy (HSMN). In addition, those pathological neurofilament accumulations are known in α-synuclein in Parkinson’s disease (PD), Aβ and tau in Alzheimer’s disease (AD), polyglutamine in CAG trinucleotide repeat disorders, superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP43), neuronal FUS proteins, optineurin (OPTN), ubiquilin 2 (UBQLN2), and dipeptide repeat protein (DRP) in amyotrophic lateral sclerosis (ALS). When axon damage occurs in central nervous disorders, neurofilament proteins are released and delivered into cerebrospinal fluid (CSF), which are then circulated into blood. New quantitative analyses and assay techniques are well-developed for the detection of neurofilament proteins, particularly NF-L and the phosphorylated NF-H (pNF-H) in CSF and serum. This review discusses the potential of using peripheral blood NF quantities and evaluating the severity of damage in the nervous system. Intermediate filaments could be promising biomarkers for evaluating disease progression in different nervous system disorders.

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Molofsky, Ari B., Brenda G. Byrne, Natalie N. Whitfield, Cressida A. Madigan, Etsu T. Fuse, Kazuhiro Tateda und Michele S. Swanson. „Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection“. Journal of Experimental Medicine 203, Nr. 4 (10.04.2006): 1093–104. http://dx.doi.org/10.1084/jem.20051659.

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To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1β secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection.

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George, Margaret D., Robert N. Wine, Brad Lackford, Grace E. Kissling, Steven K. Akiyama, Kenneth Olden und John D. Roberts. „p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion“. Biochemistry and Cell Biology 91, Nr. 6 (Dezember 2013): 404–18. http://dx.doi.org/10.1139/bcb-2013-0013.

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Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to the spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV, as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia, whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading, and that this association can be regulated by factors in the tumor microenvironment.

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Bridge, Dacie R., Karen H. Martin, Elizabeth R. Moore, Wendy M. Lee, James A. Carroll, Claudia L. Rocha und 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, Nr. 9 (11.06.2012): 3049–64. http://dx.doi.org/10.1128/iai.00231-12.

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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.

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Dale, Jeffrey M., und Michael L. Garcia. „Neurofilament Phosphorylation during Development and Disease: Which Came First, the Phosphorylation or the Accumulation?“ Journal of Amino Acids 2012 (18.04.2012): 1–10. http://dx.doi.org/10.1155/2012/382107.

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Posttranslational modification of proteins is a ubiquitous cellular mechanism for regulating protein function. Some of the most heavily modified neuronal proteins are cytoskeletal proteins of long myelinated axons referred to as neurofilaments (NFs). NFs are type IV intermediate filaments (IFs) that can be composed of four subunits, neurofilament heavy (NF-H), neurofilament medium (NF-M), neurofilament light (NF-L), and α-internexin. Within wild type axons, NFs are responsible for mediating radial growth, a process that determines axonal diameter. NFs are phosphorylated on highly conserved lysine-serine-proline (KSP) repeats located along the C-termini of both NF-M and NF-H within myelinated axonal regions. Phosphorylation is thought to regulate aspects of NF transport and function. However, a key pathological hallmark of several neurodegenerative diseases is ectopic accumulation and phosphorylation of NFs. The goal of this review is to provide an overview of the posttranslational modifications that occur in both normal and diseased axons. We review evidence that challenges the role of KSP phosphorylation as essential for radial growth and suggests an alternative role for NF phosphorylation in myelinated axons. Furthermore, we demonstrate that regulation of NF phosphorylation dynamics may be essential to avoiding NF accumulations.

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