scholarly journals Interaction of Theiler’s Virus with Intermediate Filaments of Infected Cells

1998 ◽  
Vol 72 (12) ◽  
pp. 9553-9560 ◽  
Author(s):  
Patrick Nédellec ◽  
Patrick Vicart ◽  
Christine Laurent-Winter ◽  
Cécile Martinat ◽  
Marie-Christine Prévost ◽  
...  
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Close Contact ◽  
Theiler's Virus ◽  
Virus Particles ◽  
Host Cell Proteins ◽  
Encephalomyelitis Virus ◽  
Infected Cells ◽  
Main Components ◽  
Filament Network

ABSTRACT Theiler’s murine encephalomyelitis virus is a neurotropic murine picornavirus which replicates permissively and causes a cytopathic effect in the BHK-21 cell line. We examined the interactions between the GDVII and DA strains of Theiler’s virus and BHK-21 host cell proteins in a virus overlay assay. We observed binding of the virions to two proteins of approximately 60 kDa. These proteins were microsequenced and identified as desmin and vimentin, two main components of the intermediate filament network. The association between desmin or vimentin and virions was demonstrated by immunoprecipitation. Anti-desmin and anti-vimentin monoclonal antibodies precipitated GDVII or DA virions from extracts of infected BHK-21 cells. The intracellular distributions of virions and of the desmin and vimentin intermediate filaments of BHK-21 cells were investigated by two-color immunofluorescence confocal microscopy. Following infection, the intermediate filament network was rearranged into a shell-like structure which surrounded a viral inclusion. Finally, close contact between GDVII virus particles and 10-nm intermediate filaments was observed by electron microscopy.

Novel features of intermediate filament dynamics revealed by green fluorescent protein chimeras

1998 ◽  
Vol 111 (13) ◽  
pp. 1767-1778 ◽  
Author(s):  
C.L. Ho ◽  
J.L. Martys ◽  
A. Mikhailov ◽  
G.G. Gundersen ◽  
R.K. Liem
Keyword(s):  
Green Fluorescent Protein ◽  
Dynamic Behavior ◽  
Cell Lines ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Fluorescent Protein ◽  
Nih3t3 Cells ◽  
Green Fluorescent ◽  
Filament Network ◽  
Perinuclear Area

In order to study the dynamic behavior of intermediate filament networks in living cells, we have prepared constructs fusing green fluorescent protein to intermediate filament proteins. Vimentin fused to green fluorescent protein labeled the endogenous intermediate filament network. We generated stable SW13 and NIH3T3 cell lines that express an enhanced green fluorescent protein fused to the N-terminus of full-length vimentin. We were able to observe the dynamic behavior of the intermediate filament network in these cells for periods as long as 4 hours (images acquired every 2 minutes). In both cell lines, the vimentin network constantly moves in a wavy manner. In the NIH3T3 cells, we observed extension of individual vimentin filaments at the edge of the cell. This movement is dependent on microtubules, since the addition of nocodazole stopped the extension of the intermediate filaments. Injection of anti-IFA causes the redistribution or ‘collapse’ of intermediate filaments. We injected anti-IFA antibodies into NIH3T3 cells stably expressing green fluorescent protein fused to vimentin and found that individual intermediate filaments move slowly towards the perinuclear area without obvious disassembly. These results demonstrate that individual intermediate filaments are translocated during the collapse, rather than undergoing disassembly-induced redistribution. Injections of tubulin antibodies disrupt the interactions between intermediate filaments and stable microtubules and cause the collapse of the vimentin network showing that these interactions play an important role in keeping the intermediate filament network extended. The nocodazole inhibition of intermediate filament extension and the anti-IFA microinjection experiments are consistent with a model in which intermediate filaments exhibit an extended distribution when tethered to microtubules, but are translocated to the perinuclear area when these connections are severed.

Interactions between peripherin and neurofilaments in cultured cells: disruption of peripherin assembly by the NF-M and NF-H subunits

1999 ◽  
Vol 77 (1) ◽  
pp. 41-45 ◽  
Author(s):  
Jean-Martin Beaulieu ◽  
Janice Robertson ◽  
Jean-Pierre Julien
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Intermediate Filament Protein ◽  
Physiological Conditions ◽  
Filament Protein ◽  
Filament Network ◽  
Filament Type

Neurofilaments are the principal intermediate filament type expressed by neurons. They are formed by the co-assembly of three subunits: NF-L, NF-M, and NF-H. Peripherin is another intermediate filament protein expressed mostly in neurons of the peripheral nervous system. In contrast to neurofilaments, peripherin can self-assemble to establish an intermediate filament network in cultured cells. The co-expression of neurofilaments and peripherin is found mainly during development and regeneration. We used SW13 cells devoid of endogenous cytoplasmic intermediate filaments to assess the exact assembly characteristics of peripherin with each neurofilament subunit. Our results demonstrate that peripherin can assemble with NF-L. In contrast, the co-expression of peripherin with the large neurofilament subunits interferes with peripherin assembly. These results confirm the existence of interactions between peripherin and neurofilaments in physiological conditions. Moreover, they suggest that perturbations in the stoichiometry of neurofilaments can have an impact on peripherin assembly in vivo.Key words: peripherin, neurofilament, SW13 cells, intermediate filament.

scholarly journals The specificity of the interaction between α B-crystallin and desmin filaments and its impact on filament aggregation and cell viability

2013 ◽  
Vol 368 (1617) ◽  
pp. 20120375 ◽  
Author(s):  
Jayne L. Elliott ◽  
Ming Der Perng ◽  
Alan R. Prescott ◽  
Karin A. Jansen ◽  
Gijsje H. Koenderink ◽  
...  
Keyword(s):  
Cell Viability ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Transient Transfection ◽  
Muscle Pathology ◽  
Mcf7 Cells ◽  
Biophysical Techniques ◽  
Shock Proteins ◽  
Filament Network ◽  
Innate Ability

CRYAB ( α B-crystallin) is expressed in many tissues and yet the R120G mutation in CRYAB causes tissue-specific pathologies, namely cardiomyopathy and cataract. Here, we present evidence to demonstrate that there is a specific functional interaction of CRYAB with desmin intermediate filaments that predisposes myocytes to disease caused by the R120G mutation. We use a variety of biochemical and biophysical techniques to show that plant, animal and ascidian small heat-shock proteins (sHSPs) can interact with intermediate filaments. Nevertheless, the mutation R120G in CRYAB does specifically change that interaction when compared with equivalent substitutions in HSP27 (R140G) and into the Caenorhabditis elegans HSP16.2 (R95G). By transient transfection, we show that R120G CRYAB specifically promotes intermediate filament aggregation in MCF7 cells. The transient transfection of R120G CRYAB alone has no significant effect upon cell viability, although bundling of the endogenous intermediate filament network occurs and the mitochondria are concentrated into the perinuclear region. The combination of R120G CRYAB co-transfected with wild-type desmin, however, causes a significant reduction in cell viability. Therefore, we suggest that while there is an innate ability of sHSPs to interact with and to bind to intermediate filaments, it is the specific combination of desmin and CRYAB that compromises cell viability and this is potentially the key to the muscle pathology caused by the R120G CRYAB.

scholarly journals A network of transverse and longitudinal intermediate filaments is associated with sarcomeres of adult vertebrate skeletal muscle.

1983 ◽  
Vol 96 (2) ◽  
pp. 562-570 ◽  
Author(s):  
K Wang ◽  
R Ramirez-Mitchell
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Electron Microscopic ◽  
Double Ring ◽  
Short Transverse ◽  
Sds Page ◽  
Scanning Electron Microscopic ◽  
Microscopic Studies ◽  
Filament Network ◽  
Rabbit Skeletal Muscles

An extensive network of transverse and longitudinal filamentous bridges was revealed when small myofibril bundles, prepared from Triton-EGTA-treated rabbit skeletal muscles, were extracted with Kl to remove the majority of thin and thick filaments. Transmission and scanning electron microscopic studies of these salt-resistant cytoskeletal residues indicated (a) small bundles of short transverse filaments connect adjacent myofibrils by forming Z to Z and M to M bridges; (b) parallel, continuous longitudinal filaments connect the peripheries of successive Z-disks and ensheath the sarcomere. These transverse and longitudinal filaments have the characteristic morphology of intermediate filaments; (c) two rings of tightly interwoven and tangled filaments, connected laterally by short filaments, encircle each Z disk. This double-ring also encircles a weblike meshwork which penetrates the sarcomeric space. From the peripheries of these rings, transverse and longitudinal intermediate filaments emerge; and (d) a massive amount of material translocated and accumulated near Z disks during Kl extraction. The residues were fairly resistant to solubilization by urea and SDS, and complete dissolution was achieved only with guanidinium chloride. SDS PAGE indicated that the residues consisted mainly of titin, nebulin, and variable amounts of residual myosin and actin. Desmin represented only a few percent of total residual proteins; however, it may be a major component of the intermediate filament network. We suggest that the intermediate filament should be considered an integral sarcomeric component that may play important cytoskeletal roles in muscle structure and mechanics.

Inhibition of vimentin synthesis and disruption of intermediate filaments in simian virus 40-infected monkey kidney cells

1984 ◽  
Vol 4 (9) ◽  
pp. 1880-1889
Author(s):  
A Ben-Ze'ev
Keyword(s):  
Intermediate Filament ◽  
Blot Hybridization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Monkey Kidney ◽  
In Vitro Translation ◽  
Translation System ◽  
Kidney Cells ◽  
Infected Cells ◽  
Filament Network

The organization, synthesis, and phosphorylation of vimentin were studied at various times after infection of monkey kidney cells with simian virus 40. Late after infection (between 36 and 48 h postinfection) there is a dramatic reduction in vimentin synthesis that is paralleled by a specific disruption of the intermediate filament network. At the same time there is no apparent alteration of the organization or the synthesis of the actin-containing filaments and of the microtubules. The inhibition of vimentin synthesis is also reflected by the level of vimentin mRNA activity in the infected cells, as assayed in a cell-free in vitro translation system, and vimentin mRNA concentration as revealed by RNA blot hybridization to cloned vimentin cDNA. The level of vimentin phosphorylation also decreases dramatically but at a much earlier time after infection (between 14 and 24 h postinfection), when mitosis in the infected cells is blocked. Although the decrease in vimentin synthesis in simian virus 40-infected cells is paralleled by the alterations in the organization of the intermediate filament network, the phosphorylation of vimentin correlates with the cell cycle, as it does in other systems. A possible feedback control mechanism of vimentin synthesis by alterations in the organization of the intermediate filament network is discussed.

scholarly journals Inhibition of vimentin synthesis and disruption of intermediate filaments in simian virus 40-infected monkey kidney cells.

1984 ◽  
Vol 4 (9) ◽  
pp. 1880-1889 ◽  
Author(s):  
A Ben-Ze'ev
Keyword(s):  
Intermediate Filament ◽  
Blot Hybridization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Monkey Kidney ◽  
In Vitro Translation ◽  
Translation System ◽  
Kidney Cells ◽  
Infected Cells ◽  
Filament Network

The organization, synthesis, and phosphorylation of vimentin were studied at various times after infection of monkey kidney cells with simian virus 40. Late after infection (between 36 and 48 h postinfection) there is a dramatic reduction in vimentin synthesis that is paralleled by a specific disruption of the intermediate filament network. At the same time there is no apparent alteration of the organization or the synthesis of the actin-containing filaments and of the microtubules. The inhibition of vimentin synthesis is also reflected by the level of vimentin mRNA activity in the infected cells, as assayed in a cell-free in vitro translation system, and vimentin mRNA concentration as revealed by RNA blot hybridization to cloned vimentin cDNA. The level of vimentin phosphorylation also decreases dramatically but at a much earlier time after infection (between 14 and 24 h postinfection), when mitosis in the infected cells is blocked. Although the decrease in vimentin synthesis in simian virus 40-infected cells is paralleled by the alterations in the organization of the intermediate filament network, the phosphorylation of vimentin correlates with the cell cycle, as it does in other systems. A possible feedback control mechanism of vimentin synthesis by alterations in the organization of the intermediate filament network is discussed.

scholarly journals Interaction of frog virus-3 with the cytoskeleton. I. Altered organization of microtubules, intermediate filaments, and microfilaments.

1983 ◽  
Vol 96 (5) ◽  
pp. 1248-1257 ◽  
Author(s):  
K G Murti ◽  
R Goorha
Keyword(s):  
Intermediate Filaments ◽  
Virus Assembly ◽  
Cell Periphery ◽  
Frog Virus 3 ◽  
Frog Virus ◽  
Viral Genomes ◽  
Virus Particles ◽  
Microfilament Bundles ◽  
Infected Cells ◽  
Triton X

The progressive cytoskeletal alterations of frog virus 3-infected baby hamster kidney (BHK) and fathead minnow (FHM) cells were studied by immunofluorescence and electron microscopy. The virus assembly sites, which contain viral genomes and viral proteins, were detected in the cytoplasm at 4 h (FHM) or 6 h (BHK) and mature virions appeared 2 h later. When infected cells were treated with Triton X-100, the assembly sites were found in association with the cytoskeleton. In infected cells, the number of microtubules progressively decreased but a few microtubules traversing in the vicinity of the assembly sites remained intact. Early in infection, the intermediate filaments retracted from the cell periphery, delimited the forming assembly sites, and remained there throughout infection. We suggest that intermediate filaments are involved in the formation of assembly sites. In addition, the filaments either by themselves or in conjunction with microtubules may anchor the assembly sites near the nucleus. The microfilament bundles (stress fibers) disappeared with the formation of assembly sites, and late in infection many projections containing microfilaments and virus particles appeared at the cell surface. The observation suggests a role for microfilaments in virus release. Taken together, these results provide the first example of a virus-infected cell in which all three cytoskeletal filaments show profound organizational changes and suggest an active participation of the host cytoskeleton in viral functions.

scholarly journals Neurofilaments are obligate heteropolymers in vivo

1993 ◽  
Vol 122 (6) ◽  
pp. 1337-1350 ◽  
Author(s):  
MK Lee ◽  
Z Xu ◽  
PC Wong ◽  
DW Cleveland
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Dna Transfection ◽  
Tail Domain ◽  
In Vitro Assembly ◽  
Mature Neurons ◽  
Filament Network

Neurofilaments (NFs), composed of three distinct subunits NF-L, NF-M, and NF-H, are neuron-specific intermediate filaments present in most mature neurons. Using DNA transfection and mice expressing NF transgenes, we find that despite the ability of NF-L alone to assemble into short filaments in vitro NF-L cannot form filament arrays in vivo after expression either in cultured cells or in transgenic oligodendrocytes that otherwise do not contain a cytoplasmic intermediate filament (IF) array. Instead, NF-L aggregates into punctate or sheet like structures. Similar nonfilamentous structures are also formed when NF-M or NF-H is expressed alone. The competence of NF-L to assemble into filaments is fully restored by coexpression of NF-M or NF-H to a level approximately 10% of that of NF-L. Deletion of the head or tail domain of NF-M or substitution of the NF-H tail onto an NF-L subunit reveals that restoration of in vivo NF-L assembly competence requires an interaction provided by the NF-M or NF-H head domains. We conclude that, contrary to the expectation drawn from earlier in vitro assembly studies, NF-L is not sufficient to assemble an extended filament network in an in vivo context and that neurofilaments are obligate heteropolymers requiring NF-L and NF-M or NF-H.

scholarly journals A novel photoactivatable tool for intermediate filament disruption indicates a role for keratin filaments in early embryogenesis

2018 ◽  
Author(s):  
Rucha Sanghvi-Shah ◽  
Shalaka Paranjpe ◽  
Jiyeon Baek ◽  
Radek Dobrowolski ◽  
Gregory F. Weber
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cell Types ◽  
Model Systems ◽  
Intermediate Filament Proteins ◽  
Morphogenetic Movements ◽  
Cell Functions ◽  
Filament Network ◽  
Keratin Intermediate Filaments

AbstractThe significance of cytoplasmic intermediate filament proteins has previously been examined largely through various genetic approaches, including knockdown, knockout and transgenic overexpression. Few studies to date have attempted to examine the role of specifically the filamentous intermediate filament network in orchestrating various cell functions. To directly assess the role of the filamentous keratin intermediate filament network in regulation of cellular behavior, we created a PhotoActivatable disruptor of keratin Intermediate Filaments (PA-dIF). This genetically encoded construct consists of a peptide derived from the 2B2 region of Keratin 8 fused to the photosensitive LOV2 domain from Avena sativa phototropin-1. Upon 458 nm photoirradiation, PA-dIF disrupts keratin intermediate filaments in multiple species and cell types. Marked remodeling of the keratin intermediate filament network accompanies collective cellular morphogenetic movements that occur during gastrulation and neurulation in the Xenopus laevis frog embryo. Light-based activation of PA-dIF was able to disrupt keratin intermediate filaments in Xenopus cells and lead to tissue-specific disruption of morphogenetic processes. Altogether our data show a fundamental requirement for keratin intermediate filaments in orchestrating morphogenetic movements during early embryonic development that have yet to be revealed in other model systems. Moreover, our data validate the utility of a new genetically encoded photoactivatable tool for the disruption and examination of intermediate filaments.

Transient expression of the intermediate filament nestin during skeletal muscle development

1993 ◽  
Vol 106 (4) ◽  
pp. 1291-1300 ◽  
Author(s):  
T. Sejersen ◽  
U. Lendahl
Keyword(s):  
Skeletal Muscle ◽  
Transient Expression ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Muscle Development ◽  
Skeletal Muscle Development ◽  
Adult Rat ◽  
Before And After ◽  
Filament Network

It has previously been established that skeletal muscle development is accompanied by changes in the composition of intermediate filaments: vimentin is expressed predominantly in myoblasts and desmin in adult myotubes. We show that the intermediate filament transitions during muscle development are more complex, and involve a transient expression of the recently discovered intermediate filament nestin. Nestin RNA is expressed predominantly early, in a biphasic pattern, and is markedly downregulated in adult rat muscle, whereas desmin RNA becomes more abundant throughout development. Nestin protein was found up to the postnatal myotube stage, where it colocalized with desmin in Z bands. The intracellular distribution of nestin, vimentin and desmin was analysed in the human myogenic cell line G6 before and after in vitro differentiation. Despite its more distant evolutionary and structural relationship to the other two intermediate filaments, nestin formed a cytoplasmic filamentous network indistinguishable from that of desmin and vimentin, both in undifferentiated myoblasts and after differentiation to multinuclear myotubes. In conclusion, our data suggest that nestin is an integrated component of the dynamic intermediate filament network during muscle development and that nestin copolymerizes with desmin and vimentin at stages of coexpression.

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