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H. Herrmann, & J.R. Harris. New York: Plenum Press. This is a easily readable treatise on one of the spin-offs produced by research in cell biology. The molecular characterization of peculiar fibrous proteins, that is cytokeratins, including the identification of new ones thought to be degradation products by many in the field (e.g. CK2e and CK2p) and the analysis of their expression patterns immediately led to the recognition of the merit of anti-CK antibodies for routine use as cell lineage and differentiation markers in tumour pathology
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Moll R. Cytokeratins as markers of differentiation in the diagnosis of epithelial tumors. Herrmann H., Harris J.R. Subcellular Biochemistry, vol 31: Intermediate filaments. 1998;205-262 Plenum Press, New York. This is a easily readable treatise on one of the spin-offs produced by research in cell biology. The molecular characterization of peculiar fibrous proteins, that is cytokeratins, including the identification of new ones thought to be degradation products by many in the field (e.g. CK2e and CK2p) and the analysis of their expression patterns immediately led to the recognition of the merit of anti-CK antibodies for routine use as cell lineage and differentiation markers in tumour pathology.
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Moll, R.1
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T. Kreis, & R. Vale. Oxford: Oxford University Press. A lucid and concise account of the structural organization of IF proteins as deduced from the many available amino acid sequences. Despite very similar numbers of residues in the central α-helical rod domain of hard α-keratin, epidermal CKs and vimentin (311, 314 and 309), respectively, and identical numbers of residues in the helical parts (276) therein, rather different axial periods (or repeat lengths) of 47.0, 45.2 and 42.7 nm are obtained. At present, it is not clear how the minimal differences in the linker sequences connecting the four helical segments can yield such large differences unless the helical pitches of the coiled-coils are, despite identical amino acid number, different. Considering the unique 50 nm repeat length of lamins, it is postulated that four unique IF structures must exist. This might explain why the IF proteins of one assembly group do not coassemble with those of another group
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Parry D.A.D. Structural features of IF proteins. Kreis T., Vale R. Guidebook to the Cytoskeletal and Motor Proteins. 1999;285-291 Oxford University Press, Oxford. A lucid and concise account of the structural organization of IF proteins as deduced from the many available amino acid sequences. Despite very similar numbers of residues in the central α-helical rod domain of hard α-keratin, epidermal CKs and vimentin (311, 314 and 309), respectively, and identical numbers of residues in the helical parts (276) therein, rather different axial periods (or repeat lengths) of 47.0, 45.2 and 42.7 nm are obtained. At present, it is not clear how the minimal differences in the linker sequences connecting the four helical segments can yield such large differences unless the helical pitches of the coiled-coils are, despite identical amino acid number, different. Considering the unique 50 nm repeat length of lamins, it is postulated that four unique IF structures must exist. This might explain why the IF proteins of one assembly group do not coassemble with those of another group. However, it has been shown recently that a simple epithelial CK (CK8) can form bona fide IFs with the type I hair CK hHa1 [94]. Hence, the axial repeat distances might not be the ultimate determinant for compatibility of coassembly. This supports the view that the very heterogeneous group of 'hard' and 'soft' CKs might form a functional assembly group.
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Herrmann H., Aebi U. Intermediate filament assembly: temperature sensitivity and polymorphism. Cell Mol Life Sci. 55:1999;1416-1431.
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Hofmann I., Franke W.W. Heterotypic interactions and filament assembly of type I and type II cytokeratins in vitro: viscometry and determinations of relative affinities. Eur J Cell Biol. 72:1997;122-132.
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The characterization of nine functional type I human hair ('hard') cytokeratin genes within a 140-kbp domain on chromosome 17 enabled investigation of the complex cytokeratin expression in the hair-forming compartment by in situ hybridization experiments and by immunofluorescence microscopy using a panel of specific antibodies raised against unique peptides. Thus, differentiation of the terminal (anagen) scalp hair is described, for the first time, in molecular terms as a sequential expression of hair cytokeratins with regard to the acidic (type I) subfamily. Future studies, including expression patterns of the basic hair CK proteins as well as that of keratin-associated proteins, will ultimately yield a detailed insight how, in this unique cell differentiation system, the coordinated expression of the various proteins leads to such an extreme cytoskeleton formation like the hair
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Langbein L., Rogers M.A., Winter H., Praetzel S., Beckhaus U., Rackwitz H-R., Schweizer J. The catalog of human hair keratins. I. Expression of the nine type I members in the hair follicle. J Biol Chem. 274:1999;19874-19884. The characterization of nine functional type I human hair ('hard') cytokeratin genes within a 140-kbp domain on chromosome 17 enabled investigation of the complex cytokeratin expression in the hair-forming compartment by in situ hybridization experiments and by immunofluorescence microscopy using a panel of specific antibodies raised against unique peptides. Thus, differentiation of the terminal (anagen) scalp hair is described, for the first time, in molecular terms as a sequential expression of hair cytokeratins with regard to the acidic (type I) subfamily. Future studies, including expression patterns of the basic hair CK proteins as well as that of keratin-associated proteins, will ultimately yield a detailed insight how, in this unique cell differentiation system, the coordinated expression of the various proteins leads to such an extreme cytoskeleton formation like the hair.
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H. Herrmann, & J.R. Harris. New York: Plenum Press
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Leung C.L., Flores R.L., Liem R.K.H. The complexity of intermediate filaments in the nervous system. Herrmann H., Harris J.R. Subcellular Biochemistry, vol 31: Intermediate filaments. 1998;497-526 Plenum Press, New York.
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Goulielmos G., Gounari F., Remington S., Müller S., Häner M., Aebi U., Georgatos S.D. Filensin and phakinin form a novel type of beaded intermediate filaments and coassemble de novo in cultured cells. J Cell Biol. 132:1996;643-655.
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Molecular characteristics and interactions of the intermediate filament protein synemin. Interactions with α-actinin may anchor synemin-containing heterofilaments
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The cloning of avian synemin cDNA proved it to be a bona fide IF protein with a conserved, approximately 310 amino acid residue long α-helical rod domain and a 1290 amino acid residue long carboxy-terminal non-α-helical tail domain. In vitro this tail domain binds to α-actinin and therefore indicates how desmin and vimentin, which both appear to form heteropolymers with synemin, may anchor to the actin (i.e. microfilament) system. This type of linkage may play an important role in the generation of cytoskeletal integrity and, particularly in muscle cells, of contractile function
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Bellin R.M., Sernett S.W., Becker B., Ip W., Huiatt T.W., Robson R.M. Molecular characteristics and interactions of the intermediate filament protein synemin. Interactions with α-actinin may anchor synemin-containing heterofilaments. J Biol Chem. 274:1999;29493-29499. The cloning of avian synemin cDNA proved it to be a bona fide IF protein with a conserved, approximately 310 amino acid residue long α-helical rod domain and a 1290 amino acid residue long carboxy-terminal non-α-helical tail domain. In vitro this tail domain binds to α-actinin and therefore indicates how desmin and vimentin, which both appear to form heteropolymers with synemin, may anchor to the actin (i.e. microfilament) system. This type of linkage may play an important role in the generation of cytoskeletal integrity and, particularly in muscle cells, of contractile function.
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Bellin, R.M.1
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Hemken P.M., Bellin R.M., Sernett S.W., Becker B., Huiatt T.W., Robson R.M. Molecular characteristics of the novel intermediate filament protein paranemin. J Biol Chem. 272:1997;32489-32499.
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16
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Properties of the novel intermediate filament protein synemin and its identification in mammalian muscle
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For the first time, the occurrence of synemin in mammalian muscle has been demonstrated, using polyclonal antibodies raised against highly purified avian gizzard synemin. Myofibrillar Z-lines are stained in porcine skeletal and cardiac muscle in a punctate fashion as noted previously with avian muscle by Elias Lazarides' group [96]. It is also shown that synemin is perhaps the most calpain-labile substrate of muscle. The colocalization of synemin, calpain and desmin at Z-line regions suggests that some desmin IF myopathies may be caused by degradation of synemin thereby leading to disintegration of the muscle cytoskeleton. Evidently, synemin occupies the same cell regions as plectin, as both colocalize extensively with desmin. If desmin and synemin bind plectin at different sites, this would be the starting module for networking at the periphery of Z-disks
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Bilak S.R., Sernett S.W., Bilak M.M., Bellin R.M., Stromer M.H., Huiatt T.W., Robson R.M. Properties of the novel intermediate filament protein synemin and its identification in mammalian muscle. Arch Biochem Biophys. 355:1998;63-76. For the first time, the occurrence of synemin in mammalian muscle has been demonstrated, using polyclonal antibodies raised against highly purified avian gizzard synemin. Myofibrillar Z-lines are stained in porcine skeletal and cardiac muscle in a punctate fashion as noted previously with avian muscle by Elias Lazarides' group [96]. It is also shown that synemin is perhaps the most calpain-labile substrate of muscle. The colocalization of synemin, calpain and desmin at Z-line regions suggests that some desmin IF myopathies may be caused by degradation of synemin thereby leading to disintegration of the muscle cytoskeleton. Evidently, synemin occupies the same cell regions as plectin, as both colocalize extensively with desmin. If desmin and synemin bind plectin at different sites, this would be the starting module for networking at the periphery of Z-disks.
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Robson, R.M.7
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17
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A high molecular weight intermediate filament-associated protein in BHK-21 cells is nestin, a type VI intermediate filament protein
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Nestin - originally identified in neuro-epithelial stem cells and later also found to be expressed in embryonic heart, developing teeth, testes and somitic myoblasts - is shown to be expressed in various fibroblast cell lines and primary human foreskin fibroblasts. By copolymerization and cross-linking experiments, it is demonstrated that nestin forms heterodimers with vimentin and α-internexin but not with type I or type II CK proteins. Thus, similar to the two neurofilament triplet proteins NF-M and NF-H, nestin is a non-selfassembling IF protein that is able to form heteropolymers with vimentin
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Steinert P.M., Chou Y-H., Prahlad V., Parry D.A.D., Marekov L.N., Wu K.C., Jang S-I., Goldman R.D. A high molecular weight intermediate filament-associated protein in BHK-21 cells is nestin, a type VI intermediate filament protein. J Biol Chem. 274:1999;9881-9890. Nestin - originally identified in neuro-epithelial stem cells and later also found to be expressed in embryonic heart, developing teeth, testes and somitic myoblasts - is shown to be expressed in various fibroblast cell lines and primary human foreskin fibroblasts. By copolymerization and cross-linking experiments, it is demonstrated that nestin forms heterodimers with vimentin and α-internexin but not with type I or type II CK proteins. Thus, similar to the two neurofilament triplet proteins NF-M and NF-H, nestin is a non-selfassembling IF protein that is able to form heteropolymers with vimentin.
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Steinert, P.M.1
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0033555523
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Molecular parameters of type IV α-internexin and type IV-type III α-internexin-vimentin copolymer intermediate filaments
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By cross-linking experiments it is shown that vimentin and α-internexin form heterodimers in vitro that are able to assemble into copolymer IFs. The axial length of α-internexin homodimers is the same as that of α-internexin/vimentin heterodimers and vimentin homodimers; moreover the molecular alignments found in α-internexin and vimentin homopolymer IFs are the same as those of α-internexin/vimentin copolymer IFs indicating that α-internexin should also be able to integrate into pre-existing vimentin IFs in vivo. As α-internexin homodimers and α-internexin/vimentin heterodimers are more stable than vimentin homodimers, this may be of physiological importance by providing IFs with new qualities during neuronal development
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Steinert P.M., Marekov L.N., Parry D.A.D. Molecular parameters of type IV α-internexin and type IV-type III α-internexin-vimentin copolymer intermediate filaments. J Biol Chem. 274:1999;1657-1666. By cross-linking experiments it is shown that vimentin and α-internexin form heterodimers in vitro that are able to assemble into copolymer IFs. The axial length of α-internexin homodimers is the same as that of α-internexin/vimentin heterodimers and vimentin homodimers; moreover the molecular alignments found in α-internexin and vimentin homopolymer IFs are the same as those of α-internexin/vimentin copolymer IFs indicating that α-internexin should also be able to integrate into pre-existing vimentin IFs in vivo. As α-internexin homodimers and α-internexin/vimentin heterodimers are more stable than vimentin homodimers, this may be of physiological importance by providing IFs with new qualities during neuronal development.
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With respect to their tripartite domain organization, invertebrate IF proteins resemble nuclear lamins in their coil (long form) and tail domains except for the loss of the nuclear localization signal and the membrane attachment motif. Here it is shown that the in vitro assembly of authentic invertebrate IF proteins, isolated from the snail Helix pomatia and the nematode Ascaris suum, proceeds through similar stages to those observed with both vertebrate cytoplasmic IF proteins and lamins. Notably, the buffer conditions established to yield oligomers, intermediate assembly stages and mature filaments are quite diverse and distinct from physiological values (as used for vimentin assembly) and resemble those employed in cytokeratin IF assembly
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Geisler N., Schünemann J., Weber K., Häner M., Aebi U. Assembly and architecture of invertebrate cytoplasmic intermediate filaments reconcile features of vertebrate cytoplasmic and nuclear lamin-type intermediate filaments. J Mol Biol. 282:1998;601-617. With respect to their tripartite domain organization, invertebrate IF proteins resemble nuclear lamins in their coil (long form) and tail domains except for the loss of the nuclear localization signal and the membrane attachment motif. Here it is shown that the in vitro assembly of authentic invertebrate IF proteins, isolated from the snail Helix pomatia and the nematode Ascaris suum, proceeds through similar stages to those observed with both vertebrate cytoplasmic IF proteins and lamins. Notably, the buffer conditions established to yield oligomers, intermediate assembly stages and mature filaments are quite diverse and distinct from physiological values (as used for vimentin assembly) and resemble those employed in cytokeratin IF assembly.
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Svitkina T.M., Verkhovsky A.B., Borisy G.G. Plectin sidearms mediate interaction of intermediate filaments with microtubules and other components of the cytoskeleton. J Cell Biol. 135:1996;991-1007.
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Leung C.L., Sun D., Liem R.K.H. The intermediate filament protein peripherin is the specific interaction partner of mouse BPAG1-n (dystonin) in neurons. J Cell Biol. 144:1999;435-446.
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The complexity of interactions performed by the plakin BPAG1 and its isoforms in the neuronal cytoskeleton is investigated in BPAG1-null, NF-L-null and BPAG1/NF-L double-null mice and cells derived thereof. In particular, the direct biochemical demonstration of a microtubule-binding domain (M1) in BPAG1 defines these proteins as IF-, microfilament- and microtubule-associated factors thereby underscoring their importance as key players in the integration of the three cytoskeletal filament systems in vivo
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•]) but, beside its occurrence in epidermis and simple epithelia, it is part of desmosomes in non-epithelial cells of the dendritic reticulum of lymphatic follicles. This indicates that different PKPs might mediate specialized functions within the cell-type-specific cell adhesion complexes anchoring various IF systems to the plasma membrane and thereby to neighboring cells.
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During cell spreading, the targeted transport of small vimentin aggregates ('dots') to the cell periphery has been documented. These dots eventually fuse into short fibers ('squiggles'), which then also move to the cell periphery as shown in living cells employing transfection of cells with a green fluorescent protein (GFP)-tagged vimentin. While the transport depends on an intact microtubule system, the fusion event is independent of microtubules. In particular, biochemical and immunofluorescence studies revealed that a conventional kinesin-type molecule was associated with the vimentin dots and suggested that this motor protein facilitates the observed movements. As expected, microinjection of anti-kinesin antibodies into spreading cells inhibited the formation of extended IF network
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Live observation of fibroblastic cells (BHK-21), transiently transfected with a green fluorescent protein (GFP)-tagged human vimentin, revealed the dynamics of interphase vimentin IF arrays. Fluorescence recovery after photobleaching demonstrated the pronounced dynamics of individual fibers with respect to subunit exchange, with a half time of recovery of about 5 minutes, and the apparently independent relative movement of individual fibers. Short filamentous structures ('squiggles') move to the cell periphery with speeds of up to 11 μm/minute. Although destruction of the actin system does not influence this movement, in the absence of microtubules only very few squiggles keep on moving. Remarkably, shape changes of vimentin IFs (bending or straightening movements) are independent of microtubules or microfilaments but are completely arrested by metabolic inhibitors, indicating that these movements require energy
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Yoon M., Moir R.D., Prahlad V., Goldman R.D. Motile properties of vimentin intermediate filament networks in living cells. J Cell Biol. 143:1998;147-157. Live observation of fibroblastic cells (BHK-21), transiently transfected with a green fluorescent protein (GFP)-tagged human vimentin, revealed the dynamics of interphase vimentin IF arrays. Fluorescence recovery after photobleaching demonstrated the pronounced dynamics of individual fibers with respect to subunit exchange, with a half time of recovery of about 5 minutes, and the apparently independent relative movement of individual fibers. Short filamentous structures ('squiggles') move to the cell periphery with speeds of up to 11 μm/minute. Although destruction of the actin system does not influence this movement, in the absence of microtubules only very few squiggles keep on moving. Remarkably, shape changes of vimentin IFs (bending or straightening movements) are independent of microtubules or microfilaments but are completely arrested by metabolic inhibitors, indicating that these movements require energy.
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J Cell Biol
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Intermediate filaments in motion: Observations of intermediate filaments in cells using green fluorescent protein-vimentin
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In this 'video assay' seven movies are commented on with respect to the behavior of a green fluorescent protein (GFP)-vimentin chimera, stably expressed in NIH-3T3 cells, in different physiological situations. 'Mitotic cages' along with 'vimentin fountains' and other dynamic affairs can be appreciated after downloading the movies from the Internet (online version available at URL:
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Martys J.L., Ho C-L., Liem R.K.H., Gundersen G.G. Intermediate filaments in motion: observations of intermediate filaments in cells using green fluorescent protein-vimentin. Mol Biol Cell. 10:1999;1289-1295. In this 'video assay' seven movies are commented on with respect to the behavior of a green fluorescent protein (GFP)-vimentin chimera, stably expressed in NIH-3T3 cells, in different physiological situations. 'Mitotic cages' along with 'vimentin fountains' and other dynamic affairs can be appreciated after downloading the movies from the Internet (online version available at URL: http//www.molbiocell.org ).
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Microinjection of detyrosinated tubulin (Glu tubulin), stably modified to stay nonpolymerized and nontyrosinatable by iodoacetamide treatment, into mouse fibroblasts (NIH-3T3) disrupted the interaction of microtubules with intermediate filaments - as indicated by their collapse into perinuclear bundles - without, however, affecting microtubule arrays. The same effect was obtained with a 14 kDa carboxy-terminal fragment of Glu tubulin. Both reagents also inhibited the interaction of kinesin with microtubules in vitro. Therefore, the long known post-translational modification of tubulin (i.e. detyrosination) is very likely to be instrumental in the generation of stable microtubule-IF interaction and apparently overrides a possible interconnection of both systems by crossbridging molecules such as plectin. Unfortunately, this question has not been pursued in this study
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Kreitzer G., Liao G., Gundersen G.G. Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism. Mol Biol Cell. 10:1999;1105-1118. Microinjection of detyrosinated tubulin (Glu tubulin), stably modified to stay nonpolymerized and nontyrosinatable by iodoacetamide treatment, into mouse fibroblasts (NIH-3T3) disrupted the interaction of microtubules with intermediate filaments - as indicated by their collapse into perinuclear bundles - without, however, affecting microtubule arrays. The same effect was obtained with a 14 kDa carboxy-terminal fragment of Glu tubulin. Both reagents also inhibited the interaction of kinesin with microtubules in vitro. Therefore, the long known post-translational modification of tubulin (i.e. detyrosination) is very likely to be instrumental in the generation of stable microtubule-IF interaction and apparently overrides a possible interconnection of both systems by crossbridging molecules such as plectin. Unfortunately, this question has not been pursued in this study.
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