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Kevin L. Schey - One of the best experts on this subject based on the ideXlab platform.
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identification of a direct aquaporin 0 binding site in the lens specific cytoskeletal protein Filensin
Experimental Eye Research, 2017Co-Authors: Zhen Wang, Kevin L. ScheyAbstract:An interaction between the C-terminus of aquaporin-0 (AQP0) and lens beaded filament protein Filensin has been reported previously; however, the region of Filensin that is involved in the interaction has not been determined. This study is designed to identify the region of Filensin that interacts with AQP0. Chemical crosslinking coupled with mass spectrometry was used to identify the site of interaction. The protein complex was crosslinked with zero-length crosslinker: 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC). The crosslinked membrane fraction was digested by trypsin and crosslinked peptides were identified by liquid chromatography-tandem mass spectrometry. A crosslinked peptide between bovine Filensin 450-465 (VKGPKEPEPPADLYTK) and bovine AQP0 239-259 (GSRPSESNGQPEVTGEPVELK) was detected. AQP0/Filensin crosslinking was not detected in superficial young fiber cells, but increased with fiber cell age in the lens cortex. AQP0/Filensin crosslinking and Filensin truncation were observed in the same regions of the lens. This crosslinked peptide can be detected in 75 kDa gel band confirming that AQP0/Filensin crosslinking can occur between AQP0 and the Filensin C-terminal fragment. These results suggest that the AQP0 C-terminus directly interacts with the region of Filensin that is adjacent to the major truncation site and the polybasic cluster of residues in the Filensin C-terminal tail. This interaction occurs in a specific region of the lens and could only occur between AQP0 and Filensin C-terminal fragment in vivo. This interaction supports the dual roles of Filensin in the lens; roles that could be important during lens development.
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and Expression of Connexins
2016Co-Authors: Phosphorylation Of Aquaporin, Kevin L. Schey, Myosin Light Chain, Rupalatha Maddala, Tharkika Nagendran, Gustaaf G. De Ridder, Vasantha RaoAbstract:Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in b-B1-crystallin, Hsp-90, spectrin and Filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3e, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression o
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spatially directed proteomics of the human lens outer cortex reveals an intermediate filament switch associated with the remodeling zone
Investigative Ophthalmology & Visual Science, 2016Co-Authors: Jamie L Wenke, Hayes W Mcdonald, Kevin L. ScheyAbstract:PURPOSE: To quantify protein changes in the morphologically distinct remodeling zone (RZ) and adjacent regions of the human lens outer cortex using spatially directed quantitative proteomics. METHODS: Lightly fixed human lens sections were deparaffinized and membranes labeled with fluorescent wheat germ agglutinin (WGA-TRITC). Morphology directed laser capture microdissection (LCM) was used to isolate tissue from four distinct regions of human lens outer cortex: differentiating zone (DF), RZ, transition zone (TZ), and inner cortex (IC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the plasma membrane fraction from three lenses (21-, 22-, and 27-year) revealed changes in major cytoskeletal proteins including vimentin, Filensin, and phakinin. Peptides from proteins of interest were quantified using multiple reaction monitoring (MRM) mass spectrometry and isotopically-labeled internal peptide standards. RESULTS: Results revealed an intermediate filament switch from vimentin to beaded filament proteins Filensin and phakinin that occurred at the RZ. Several other cytoskeletal proteins showed significant changes between regions, while most crystallins remained unchanged. Targeted proteomics provided accurate, absolute quantification of these proteins and confirmed vimentin, periplakin, and periaxin decrease from the DF to the IC, while Filensin, phakinin, and brain acid soluble protein 1 (BASP1) increase significantly at the RZ. CONCLUSIONS: Mass spectrometry-compatible fixation and morphology directed laser capture enabled proteomic analysis of narrow regions in the human lens outer cortex. Results reveal dramatic cytoskeletal protein changes associated with the RZ, suggesting that one role of these proteins is in membrane deformation and/or the establishment of ball and socket joints in the human RZ.
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maldi imaging mass spectrometry spatially maps age related deamidation and truncation of human lens aquaporin 0
Investigative Ophthalmology & Visual Science, 2015Co-Authors: Jamie L Wenke, Kristie L Rose, Jeffrey M Spraggins, Kevin L. ScheyAbstract:The ocular lens is a transparent optical element, focusing light onto the retina for clear vision. Tight cellular packing, lack of light-scattering organelles, and a careful balance of protein and water concentration ensure lens transparency and provide focusing properties.1–3 Epithelial cells on the lens anterior surface differentiate into lens fiber cells, such that new fiber cells are continuously added to the lens cortex atop older fiber cells in the lens core, creating concentric rings that reflect cellular age.4 Lens fibers mature through a process of elongation, cellular compaction, and removal of organelles. Mature fiber cells in the core experience no cellular turnover and they are devoid of light-scattering nuclei as well as other organelles.5 Without machinery for cellular turnover or protein synthesis, long-lived cells and their contents survive for decades in the lens.6 Moreover, aging lens proteins are subject to modification over time. Some posttranslational modifications (PTMs), like phosphorylation, are programmed to occur in a specific region of the lens, while truncation accumulates in a time-dependent manner with age.7 In the lens, PTMs may alter protein function by modulating existing protein function when no new protein is produced. One essential, yet extensively modified, protein is the lens major intrinsic protein (MIP), or aquaporin-0 (AQP0). Aquaporin-0 is a dual-function water channel and adhesion molecule comprising approximately 50% of lens membrane protein.8 The central pore of each AQP0 monomer shuttles water across the plasma membrane.9 Aquaporin-0 also is involved in junction formation and cell-to-cell adhesion.10–16 Mutations or loss of AQP0 lead to cataract, highlighting the importance of this protein for lens function.17–28 Aquaporin-0 is a 28 kD protein comprised of six transmembrane domains and an intracellular C-terminal tail. The highly modified tail regulates AQP0 permeability via calmodulin binding.29–31 This region of AQP0 also binds cytoskeletal proteins, including Filensin and phakinin,32 and may have a role in establishing fiber cell organization. Numerous PTMs decorate the AQP0 C-terminus over time, some of which have been functionally characterized. Phosphorylation of Ser-235 reduces the binding affinity of calmodulin, thereby boosting the permeability of the channel.31,33,34 Cleavage of the C-terminal tail removes protein–protein binding regions and may encourage AQP0 to form cell–cell junctions.7,13 More recently, fatty acid modifications were discovered on lysine 238 and on the N-terminal methionine, which may target the protein to detergent-resistant lipid raft regions of the membrane.35,36 Deamidation and isomerization of asparagine residues have been reported but have not been fully characterized.37,38 The localization of these PTMs within the lens can provide clues of the changing physiological role of AQP0 over time. Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) has been used to map the spatial localization of modified AQP0 within the lens.35,39 In MALDI IMS, proteins are detected by their mass-to-charge (m/z) value and each m/z value can be mapped across a tissue section with intensity encoded as a heat map.40 Protein modifications are characterized by specific shifts in m/z values. Previous MALDI experiments showed the localization of truncated and lipid-modified AQP0.35,41 Although informative, these molecular images captured a single time point in the complex aging process. Furthermore, the spatial distribution and accumulation of deamidated AQP0 is unknown. The aim of this study was to characterize the localization of modified AQP0 in human lens from birth through middle age using MALDI IMS. We optimized methods to image modified AQP0, revealing unprecedented images of deamidated and truncated forms of the functionally-important AQP0 C-terminal tail. Understanding the localization of these modifications in young or old fiber cells can provide clues on how AQP0 function changes during development, aging, and cataract formation.
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The C Terminus of Lens Aquaporin 0 Interacts with the Cytoskeletal Proteins Filensin and CP49
2013Co-Authors: Kristie Lindsey M. Rose, Roy A. Quinlan, Kevin L. Schey, Alan R Prescott, Robert G Gourdie, Rosalie K Crouch, Purpose AquaporinAbstract:protein in the lens, is a water-permeable channel, has a role in fiber cell adhesion, and is essential for fiber cell structure and organization. The purpose of this study was to identify proteins that interact with the C terminus of AQP0, by using a proteomics approach, and thus further elucidate the role of AQP0 in the human lens. METHODS. AQP0 C-terminal peptides and AQP0 antibody affinity chromatography were used for affinity purification of interacting human lens proteins. Purified proteins were digested with trypsin, analyzed by liquid chromatography (LC)-tandem mass spectrometry and identified after database searching and manual examination of the mass spectral data. Colocalization of AQP0 with Filensin and CP49, two proteins identified after mass spectrometric analysis, were examined by immunoconfocal and immunoelectron microscopy of lens sections
Paul G. Fitzgerald - One of the best experts on this subject based on the ideXlab platform.
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cp49 and Filensin intermediate filaments are essential for formation of cold cataract
Molecular Vision, 2020Co-Authors: Xi Liu, Paul G. Fitzgerald, Chunhong Xia, Jessica Wang, Xiaohua GongAbstract:Author(s): Li, Yuxing; Liu, Xi; Xia, Chun-Hong; FitzGerald, Paul G; Li, Rachel; Wang, Jessica; Gong, Xiaohua | Abstract: PurposeTo investigate the molecular and cellular mechanisms of cataract induced by cold temperatures in young lenses of wild-type C57BL/6J (B6), wild-type 129SvJae (129), and Filensin knockout (KO) mice. To determine how lens intermediate filament proteins, Filensin (BFSP1) and CP49 (BFSP2), are involved in the formation of cold cataract.MethodsThe formation of cold cataract was examined in enucleated lenses at different temperatures and was imaged under a dissecting microscope. Lens vibratome sections were prepared, immunostained with different antibodies and fluorescent probes, and then imaged with a laser confocal microscope to evaluate the protein distribution and the membrane and cytoskeleton structures in the lens fibers.ResultsPostnatal day 14 (P14) wild-type B6 lenses showed cataracts dependent on cold temperatures in interior fibers about 420-875 µm (zone III) and 245-875 µm (zone II and zone III) from the lens surface, under 25 °C and 4 °C, respectively. In contrast, wild-type 129 (with CP49 gene deletion) and Filensin KO (on the B6 background) lenses did not have cold cataracts at 25 °C but displayed a reduced cold cataract, especially in zone III, at 4 °C. Immunofluorescent staining data revealed that CP49 and Filensin proteins were uniformly distributed in fiber cell cytosols without cold cataracts but accumulated or aggregated in the cell boundaries of the fibers where cold cataracts appeared.ConclusionsCP49 and Filensin are important components for the formation of cold cataract in young B6 mouse lenses. Accumulated or aggregated CP49 and Filensin beaded intermediate filaments in fiber cell boundaries might directly or indirectly contribute to the light scattering of cold cataract. Cold cataract in zone II is independent of beaded intermediate filaments. CP49 and Filensin intermediate filaments and other lens proteins probably form distinct high molecular organizations to regulate lens transparency in interior fibers.
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expression of the type vi intermediate filament proteins cp49 and Filensin in the mouse lens epithelium
Molecular Vision, 2016Co-Authors: Paul G. Fitzgerald, Ning Sun, Brad Shibata, John F HessAbstract:Purpose The differentiated lens fiber cell assembles a filamentous cytoskeletal structure referred to as the beaded filament (BF). The BF requires CP49 (bfsp2) and Filensin (bfsp1) for assembly, both of which are highly divergent members of the large intermediate filament (IF) family of proteins. Thus far, these two proteins have been reported only in the differentiated lens fiber cell. For this reason, both proteins have been considered robust markers of fiber cell differentiation. We report here that both proteins are also expressed in the mouse lens epithelium, but only after 5 weeks of age.
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Resisting the Effects of Aging: A Function for the Fiber Cell Beaded Filament
2013Co-Authors: Kyoung Hye Yoon, Tom Blankenship, Bradley Shibata, Paul G. FitzgeraldAbstract:PURPOSE. The beaded filament is a cytoskeletal structure that has been found only in the lens fiber cell. It includes phakosin and Filensin, two divergent members of the intermediate filament family of proteins that are also unique to the fiber cell. The authors sought to determine what function the beaded filament fulfills in the lens. METHODS. Light microscopy and electron microscopy were used to characterize structural changes that occurred in previously generated phakosin and Filensin knockout mice. Immunocytochemistry and electron microscopy were used to define the distribution of phakosin, Filensin, and beaded filaments. RESULTS. In phakosin and Filensin knockout mice, initial lens development and the early phases of fiber cell differentiation proceed in a manner largely indistinguishable from that of wild type. Fiber cells elongate, undergo organelle elimination, and
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Targeted deletion of the lens fiber cellspecific intermediate filament protein Filensin
2013Co-Authors: Azita Alizadeh, Teri Seeberger, John Clark, John Hess, Tom Blankenship, Paul G. FitzgeraldAbstract:PURPOSE. To determine the function of the lens fiber cell– specific cytoskeletal protein, Filensin, in lens biology. METHODS. Targeted genomic deletion was used to delete exon 1 and the transcriptional start site of the Filensin gene. Resultant chimeric animals were bred to homozygosity for the mutant allele. These animals were outbred to mice bearing the wild-type CP49 alleles to eliminate the mutant CP49 gene carried by the 129 strain of mice. Animals homozygous for the mutated Filensin gene and wild-type CP49 gene were compared with wild-type and heterozygous animals by Northern and Western blot analyses, light and electron microscopy, and slit lamp microscopy. RESULTS. Disruption of the Filensin gene successfully blocked production of Filensin mRNA, reduced levels of Filensin’s assembly partner CP49, and prevented the assembly of beaded filaments. Despite the absence of beaded filaments, lenses did not show obvious changes in fetal development, nor in the differentiation of epithelial cells into mature fiber cells, as judged by light microscopic analysis. Filensin knockouts began to show evidence of light-scattering by 2 months and worsened with age. Heterozygous animals exhibited an intermediate phenotype, showing a reduction in Filensin transcript and moderate light-scattering at 5 months. CONCLUSIONS. The lens fiber cell–specific intermediate filament protein Filensin is essential for beaded filament assembly. However, although beaded filaments are not needed for normal lens fetal development or fiber cell differentiation, they appear to be necessary for the long-term maintenance of optical clarity. The mechanism by which the absence of Filensin and the beaded filament affects optical clarity has yet to be defined. (Invest Ophthalmol Vis Sci. 2003;44:5252–5258) DOI
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Bovine Filensin Possesses Primary and Secondary Structure Similarity to Intermediate Filament Proteins
2013Co-Authors: Fotini Gounari, Paul G. Fitzgerald, Andreas Merdes, Christos A Ouzounis, John Hess, Roy Quinlan, Spyros D GeorgatosAbstract:Abstract. The eDNA coding for calf Filensin, a membrane-associated protein of the lens fiber cells, has been cloned and sequenced. The predicted 755-amino acid-long open reading frame shows primary and secondary structure similarity to intermediate filament (IF) proteins. Filensin can be divided into an NH2-terminal domain (head) of 38 amino acids, a middle domain (rod) of 279 amino acids, and a COOH-terminal domain (tail) of 438 amino acids. The head domain contains a di-arginine/aromatic amino acid motif which is also found in the head domains of various intermediate filament proteins and includes a potential protein kinase A phosphorylation site. By multiple alignment to all known IF protein sequences, the Filensin rod, which is the shortest among IF proteins, can be subdivide
Spyros D Georgatos - One of the best experts on this subject based on the ideXlab platform.
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Filensin and Phakinin Form a Novel Type of Beaded Intermediate Filaments and Coassemble De Novo in Cultured Cells
2013Co-Authors: George Goulielmos, Fotini Gounari, Susann Remington, Shirley Miiller Markus H/iner, Ueli Aebi, Spyros D GeorgatosAbstract:Abstract. The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beadedchain filaments " (BFs). BFs consist of Filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified Filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that Filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization
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The 47-kD Lens-specific Protein Phakinin Is a Tailless Intermediate Filament Protein and an Assembly Partner of Filensin
2013Co-Authors: Andreas Mercies, Fotini Gounari, Spyros D GeorgatosAbstract:Abstract. In previous studies we have characterized a lens-specific intermediate filament (IF) protein, termed Filensin. Filensin does not self-assemble into regular IFs but is known to associate with another 47-kD lensspecific protein which has been suggested to represent its assembly partner. To address this possibility, we cloned and sequenced the cDNA coding for the bovine 47-kD protein which we have termed phakinin (from the greek ~baro ~ = phakos = lens). The predicted sequence comprises 406 amino acids and shows significant similarity (31.3 % identity over 358 residues) to type I cytokeratins. Phakinin possesses a 95-residue, non-helical domain (head) and a 311 amino acid long a-helical domain punctuated with heptad repeats (rod). Similar to cytokeratin 19, phakini
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Bovine Filensin Possesses Primary and Secondary Structure Similarity to Intermediate Filament Proteins
2013Co-Authors: Fotini Gounari, Paul G. Fitzgerald, Andreas Merdes, Christos A Ouzounis, John Hess, Roy Quinlan, Spyros D GeorgatosAbstract:Abstract. The eDNA coding for calf Filensin, a membrane-associated protein of the lens fiber cells, has been cloned and sequenced. The predicted 755-amino acid-long open reading frame shows primary and secondary structure similarity to intermediate filament (IF) proteins. Filensin can be divided into an NH2-terminal domain (head) of 38 amino acids, a middle domain (rod) of 279 amino acids, and a COOH-terminal domain (tail) of 438 amino acids. The head domain contains a di-arginine/aromatic amino acid motif which is also found in the head domains of various intermediate filament proteins and includes a potential protein kinase A phosphorylation site. By multiple alignment to all known IF protein sequences, the Filensin rod, which is the shortest among IF proteins, can be subdivide
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the mouse Filensin gene structure and evolutionary relation to other intermediate filament genes
FEBS Letters, 1997Co-Authors: Fotini Gounari, Spyros D Georgatos, Niki Karagianni, Antoaneta Mincheva, Peter Lichter, Volker SchirrmacherAbstract:Filensin and phakinin are two lens-specific members of the intermediate filament (IF) superfamily of proteins. They coassemble to form a beaded submembraneous filamentous network, the beaded filaments (BFs). The low sequence homology and differences in assembly compared to other IF proteins do not allow their classification in any of the five IF subgroups. The organization of the phakinin gene exon/intron boundaries provides evidence that this partner may be sharing a common origin with type I cytokeratin genes. Here we report the molecular cloning, sequence and characterization of the mouse Filensin gene. The Filensin gene consists of 8 exons and 7 introns, with 6 introns interrupting its rod domain in a highly conserved manner characteristic of type III IF genes, like vimentin, desmin, or peripherin. Of the two tail domain exons the one adjacent to the rod domain, compares to exon 7 of the non-neuronal cytoplasmic IF gene of helix aspersa and to the lamin region bridging the end of the rod domain to the nuclear localization signal. Altogether, these observations indicate that the lens beaded filaments form an independent class of IF.
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Filensin and phakinin form a novel type of beaded intermediate filaments and coassemble de novo in cultured cells
Journal of Cell Biology, 1996Co-Authors: George N Goulielmos, Fotini Gounari, S Remington, Ueli Aebi, Shirley A Muller, Markus Haner, Spyros D GeorgatosAbstract:The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beaded-chain filaments" (BFs). BFs consist of Filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified Filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that Filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization, visualization of unfixed and unstained specimens by scanning transmission electron microscopy (STEM) reveals the the existence of a central filament which is decorated by regularly spaced 12-15-nm-diam beads. Our data suggest that the filamentous core is composed of phakinin, which exhibits a tendency to self-assemble into filament bundles, whereas the beads contain Filensin/phakinin hetero-oligomers. Filensin and phakinin copolymerize and form filamentous structures when expressed transiently in cultured cells. Experiments in IF-free SW13 cells reveal that coassembly of the lens-specific proteins in vivo does not require a preexisting IF system. In epithelial MCF-7 cells de novo forming filaments appear to grow from distinct foci and organize as thick, fibrous laminae which line the plasma membrane and the nuclear envelope. However, filament assembly in CHO and SV40-transformed lens-epithelial cells (both of which are fibroblast-like) yields radial networks which codistribute with the endogenous vimentin IFs. These observations document that the filaments formed by lens-specific IF proteins are structurally distinct from ordinary cytoplasmic IFs. Furthermore, the results suggest that the spatial arrangement of Filensin/phakinin filaments in vivo is subject to regulation by host-specific factors. These factors may involve cytoskeletal networks (e.g., vimentin IFs) and/or specific sites associated with the cellular membranes.
Roy A. Quinlan - One of the best experts on this subject based on the ideXlab platform.
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The C Terminus of Lens Aquaporin 0 Interacts with the Cytoskeletal Proteins Filensin and CP49
2013Co-Authors: Kristie Lindsey M. Rose, Roy A. Quinlan, Kevin L. Schey, Alan R Prescott, Robert G Gourdie, Rosalie K Crouch, Purpose AquaporinAbstract:protein in the lens, is a water-permeable channel, has a role in fiber cell adhesion, and is essential for fiber cell structure and organization. The purpose of this study was to identify proteins that interact with the C terminus of AQP0, by using a proteomics approach, and thus further elucidate the role of AQP0 in the human lens. METHODS. AQP0 C-terminal peptides and AQP0 antibody affinity chromatography were used for affinity purification of interacting human lens proteins. Purified proteins were digested with trypsin, analyzed by liquid chromatography (LC)-tandem mass spectrometry and identified after database searching and manual examination of the mass spectral data. Colocalization of AQP0 with Filensin and CP49, two proteins identified after mass spectrometric analysis, were examined by immunoconfocal and immunoelectron microscopy of lens sections
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the c terminus of lens aquaporin 0 interacts with the cytoskeletal proteins Filensin and cp49
Investigative Ophthalmology & Visual Science, 2006Co-Authors: Kristie L Rose, Roy A. Quinlan, Alan R Prescott, Robert G Gourdie, Rosalie K Crouch, Kevin L. ScheyAbstract:PURPOSE. Aquaporin 0 (AQP0), the most abundant membrane protein in the lens, is a water-permeable channel, has a role in fiber cell adhesion, and is essential for fiber cell structure and organization. The purpose of this study was to identify proteins that interact with the C terminus of AQP0, by using a proteomics approach, and thus further elucidate the role of AQP0 in the human lens. METHODS. AQP0 C-terminal peptides and AQP0 antibody affinity chromatography were used for affinity purification of interacting human lens proteins. Purified proteins were digested with trypsin, analyzed by liquid chromatography (LC)-tandem mass spectrometry and identified after database searching and manual examination of the mass spectral data. Colocalization of AQP0 with Filensin and CP49, two proteins identified after mass spectrometric analysis, were examined by immunoconfocal and immunoelectron microscopy of lens sections. RESULTS. The proteomics approach used to identify affinitypurified proteins revealed the lens-specific intermediate filament proteins Filensin and CP49. With immunoconfocal microscopy, regions of colocalization of AQP0 with Filensin and CP49 at the fiber cell plasma membrane in the lens cortex were defined. Immunoelectron microscopy confirmed that Filensin and AQP0 were present in the same membrane compartments. CONCLUSIONS. These studies suggest a novel interaction between an aquaporin water channel and intermediate filaments, an interaction through which AQP0 may maintain lens fiber cell shape and organization. (Invest Ophthalmol Vis Sci. 2006; 47:1562‐1570) DOI:10.1167/iovs.05-1313
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localization of two conserved cis acting enhancer regions for the Filensin gene promoter that direct lens specific expression
Experimental Eye Research, 2002Co-Authors: Shigeo Masaki, Satoshi Yonezawa, Roy A. QuinlanAbstract:Filensin is a unique eye lens intermediate filament protein, and a major component of 'beaded filament' cytoskeletal network. Expression is restricted to lens fiber cells and is believed to be functionally important for lens development by maintaining lens fiber cell shape conformation and lens transparency. The mouse Filensin gene promoter core was identified by the promoter activity analysis and found to cover the region from -56 to +4. A reporter vector driven by the Filensin promoter core was constructed and DNA fragments derived from the 5'-flanking regions of the gene were cloned into the vector to identify putative cis -acting promoter enhancing activities. The activity of these constructs was monitored by transfection into the chicken embryonic lens cell culture system. Two fragments were identified with cis -acting enhancer activity that re-conferred the lens fiber cell specific expression of the promoter. The first was a 1.7-kbp region located 7.5 kbp upstream from the transcriptional start point while a second 2.1-kbp region was found adjacent to the promoter core. Both fragments specifically directed the lens cell specific expression of only the Filensin promoter and not other heterologous promoters. Comparison of 10 kbp of human and mouse 5'-upstream sequences of the Filensin promoter revealed two highly conserved sequences that corresponded in their spacing to the putative enhancer regions. These studies have identified the first conserved regulatory sequences important in the lens specific regulation of the Filensin gene.
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identification and functional analysis of the mouse lens Filensin gene promoter
Gene, 1998Co-Authors: Shigeo Masaki, Roy A. Quinlan, Satoshi Yonezawa, Yusuke Kamachi, Hisato KondohAbstract:Filensin (also called CP94; CP95; CP97; 115kDa protein) is a component of the lens-specific beaded filament which is believed to be functionally important in lens fiber cell differentiation and in maintaining lens fiber cell conformation and transparency. A 17.2kb fragment containing the 5'-upstream sequence of the Filensin gene was isolated. S1-mapping analysis determined the transcription start point (tsp; +1) which locates at 94base pairs upstream from the initiating ATG on the Filensin gene. In addition to a major tsp, a minor tsp (-136) was observed. DNA sequence of the fragment around the tsp (-2144 to +155) was identified. Analysis of the DNA sequence of the promoter region around tsp revealed two motifs with sequence homology to Sox2 and Maf recognition sequences in addition to one GATA-1 site, two Sp1 binding sites, and three AP-2 binding motifs. No TATA-box or CCAAT-motif was found around the tsp region. A series of sequentially deleted fragments of (-2144 to +40) were fused to firefly luciferase reporter plasmid pGL2 and tested for activity in chicken embryonic lens explants. A minimal promoter region for mouse Filensin of (-70 to +40) was identified. The lens-specific promoter activity was detected using lens explants cultured within 12h after dissection. The activity was remarkably enhanced by culture in the presence of 5ng/ml of basic fibroblast growth factor. Each one of the Sp1 and AP-2 binding motifs was localized to the fragment of (-27 to +40) using electrophoretic mobility shift assays. These are the first data to identify the basic elements to the 5'-upstream sequences of the Filensin gene, namely the tsp and the minimal Filensin promoter.
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gene structure and sequence comparisons of the eye lens specific protein Filensin from rat and mouse implications for protein classification and assembly
Gene, 1997Co-Authors: Shigeo Masaki, Roy A. QuinlanAbstract:Abstract The full length cDNA sequences of rat and mouse Filensin are presented, as well as the structure of the rat Filensin gene. This gene spanned 31 kb and included seven introns. The first six introns were conserved in position and phase with those found in the intermediate filament (IF) protein genes of the type II (type II keratin), type III (vimentin) and type V (lamin). The last intron of the Filensin was unique. As none of the Filensin intron positions coincided with those unique to type I, II or IV genes, it appears that Filensin is most similar to type III genes. Comparison of the deduced amino acid sequences for rat and mouse Filensin with those of cow and chick, and with other species of IF proteins, indicated the C-terminal non- α -helical tail domain of Filensin to be one of the most divergent yet found in the vertebrate IF family. The tail domain had three conserved regions which are interrupted with two regions with lower identity. Two motifs, (1) PGDVPDGxxISKAF; and (2) KVEVVESIEKxxxxxIQTYEETxxIVET, were identified as sequences which were particularly highly conserved across species. Coassembly studies using CP49 and a physiologically derived 53 kDa-fragment of Filensin showed the motif (2) was not required for filament assembly in vitro. These data strengthen the view that the C-terminal non- α -helical domain of Filensin contributes in more than one way to Filensin function in the lens.
Fotini Gounari - One of the best experts on this subject based on the ideXlab platform.
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Filensin and Phakinin Form a Novel Type of Beaded Intermediate Filaments and Coassemble De Novo in Cultured Cells
2013Co-Authors: George Goulielmos, Fotini Gounari, Susann Remington, Shirley Miiller Markus H/iner, Ueli Aebi, Spyros D GeorgatosAbstract:Abstract. The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beadedchain filaments " (BFs). BFs consist of Filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified Filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that Filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization
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The 47-kD Lens-specific Protein Phakinin Is a Tailless Intermediate Filament Protein and an Assembly Partner of Filensin
2013Co-Authors: Andreas Mercies, Fotini Gounari, Spyros D GeorgatosAbstract:Abstract. In previous studies we have characterized a lens-specific intermediate filament (IF) protein, termed Filensin. Filensin does not self-assemble into regular IFs but is known to associate with another 47-kD lensspecific protein which has been suggested to represent its assembly partner. To address this possibility, we cloned and sequenced the cDNA coding for the bovine 47-kD protein which we have termed phakinin (from the greek ~baro ~ = phakos = lens). The predicted sequence comprises 406 amino acids and shows significant similarity (31.3 % identity over 358 residues) to type I cytokeratins. Phakinin possesses a 95-residue, non-helical domain (head) and a 311 amino acid long a-helical domain punctuated with heptad repeats (rod). Similar to cytokeratin 19, phakini
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Bovine Filensin Possesses Primary and Secondary Structure Similarity to Intermediate Filament Proteins
2013Co-Authors: Fotini Gounari, Paul G. Fitzgerald, Andreas Merdes, Christos A Ouzounis, John Hess, Roy Quinlan, Spyros D GeorgatosAbstract:Abstract. The eDNA coding for calf Filensin, a membrane-associated protein of the lens fiber cells, has been cloned and sequenced. The predicted 755-amino acid-long open reading frame shows primary and secondary structure similarity to intermediate filament (IF) proteins. Filensin can be divided into an NH2-terminal domain (head) of 38 amino acids, a middle domain (rod) of 279 amino acids, and a COOH-terminal domain (tail) of 438 amino acids. The head domain contains a di-arginine/aromatic amino acid motif which is also found in the head domains of various intermediate filament proteins and includes a potential protein kinase A phosphorylation site. By multiple alignment to all known IF protein sequences, the Filensin rod, which is the shortest among IF proteins, can be subdivide
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the mouse Filensin gene structure and evolutionary relation to other intermediate filament genes
FEBS Letters, 1997Co-Authors: Fotini Gounari, Spyros D Georgatos, Niki Karagianni, Antoaneta Mincheva, Peter Lichter, Volker SchirrmacherAbstract:Filensin and phakinin are two lens-specific members of the intermediate filament (IF) superfamily of proteins. They coassemble to form a beaded submembraneous filamentous network, the beaded filaments (BFs). The low sequence homology and differences in assembly compared to other IF proteins do not allow their classification in any of the five IF subgroups. The organization of the phakinin gene exon/intron boundaries provides evidence that this partner may be sharing a common origin with type I cytokeratin genes. Here we report the molecular cloning, sequence and characterization of the mouse Filensin gene. The Filensin gene consists of 8 exons and 7 introns, with 6 introns interrupting its rod domain in a highly conserved manner characteristic of type III IF genes, like vimentin, desmin, or peripherin. Of the two tail domain exons the one adjacent to the rod domain, compares to exon 7 of the non-neuronal cytoplasmic IF gene of helix aspersa and to the lamin region bridging the end of the rod domain to the nuclear localization signal. Altogether, these observations indicate that the lens beaded filaments form an independent class of IF.
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Filensin and phakinin form a novel type of beaded intermediate filaments and coassemble de novo in cultured cells
Journal of Cell Biology, 1996Co-Authors: George N Goulielmos, Fotini Gounari, S Remington, Ueli Aebi, Shirley A Muller, Markus Haner, Spyros D GeorgatosAbstract:The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beaded-chain filaments" (BFs). BFs consist of Filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified Filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that Filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization, visualization of unfixed and unstained specimens by scanning transmission electron microscopy (STEM) reveals the the existence of a central filament which is decorated by regularly spaced 12-15-nm-diam beads. Our data suggest that the filamentous core is composed of phakinin, which exhibits a tendency to self-assemble into filament bundles, whereas the beads contain Filensin/phakinin hetero-oligomers. Filensin and phakinin copolymerize and form filamentous structures when expressed transiently in cultured cells. Experiments in IF-free SW13 cells reveal that coassembly of the lens-specific proteins in vivo does not require a preexisting IF system. In epithelial MCF-7 cells de novo forming filaments appear to grow from distinct foci and organize as thick, fibrous laminae which line the plasma membrane and the nuclear envelope. However, filament assembly in CHO and SV40-transformed lens-epithelial cells (both of which are fibroblast-like) yields radial networks which codistribute with the endogenous vimentin IFs. These observations document that the filaments formed by lens-specific IF proteins are structurally distinct from ordinary cytoplasmic IFs. Furthermore, the results suggest that the spatial arrangement of Filensin/phakinin filaments in vivo is subject to regulation by host-specific factors. These factors may involve cytoskeletal networks (e.g., vimentin IFs) and/or specific sites associated with the cellular membranes.