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.

Intermediate filament interactions can be altered by HSP27 and alphaB-crystallin

1999 ◽  
Vol 112 (13) ◽  
pp. 2099-2112 ◽  
Author(s):  
M.D. Perng ◽  
L. Cairns ◽  
P. van den IJssel ◽  
A. Prescott ◽  
A.M. Hutcheson ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Network Formation ◽  
Specific Activity ◽  
Small Heat Shock Proteins ◽  
In Vitro Assays ◽  
Mcf7 Cells ◽  
Shock Proteins

HSP27 and alphaB-crystallin are both members of the small heat shock protein family. alphaB-crystalllin has been proposed to modulate intermediate filaments and recently a mutation in alphaB-crystallin has been identified as the genetic basis of desmin related myopathy. This disease is characterised in its pathology by aggregates of intermediate filaments associated with alphaB-crystallin. Here we report that HSP27 like alphaB-crystallin is associated with glial fibrillary acidic protein and vimentin intermediate filament networks in unstressed U373MG astrocytoma cells. HSP27 is also associated with keratin filaments in MCF7 cells, indicating that this association is not restricted to a particular intermediate filament type. The association of sHSPs with both the soluble and filamentous intermediate filament fractions of U373 cells was demonstrated biochemically. Heat shock or drug treatments induced a co-collapse of intermediate filaments and associated small heat shock proteins. These data show that the presence of HSP27 or alphaB-crystallin could not prevent filament collapse and suggest that the purpose of this association is more than just filament binding. Indeed, in U373MG cells the intermediate filament association with small heat shock proteins is similar to that observed for another protein chaperone, HSC70. In order to discern the effect of different chaperone classes on intermediate filament network formation and maintenance, several in vitro assays were assessed. Of these, falling ball viscometry revealed a specific activity of small heat shock proteins compared to HSC70 that was apparently inactive in this assay. Intermediate filaments form a gel in the absence of small heat shock proteins. In contrast, inclusion of alphaB-crystallin or HSP27 prevented gel formation but not filament assembly. The transient transfection of GFAP into MCF7 cells was used to show that the induction of a completely separate network of intermediate filaments resulted in the specific association of the endogenous HSP27 with these new GFAP filaments. These data lead us to propose that one of the major functions of the association of small heat shock proteins with intermediate filaments is to help manage the interactions that occur between filaments in their cellular networks. This is achieved by protecting filaments against those non-covalent interactions that result when they come into very close proximity as seen from the viscosity experiments and which have the potential to induce intermediate filament aggregation as seen in some disease pathologies.

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 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.

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.

scholarly journals The dynamic state of heat shock proteins in chicken embryo fibroblasts.

1986 ◽  
Vol 103 (4) ◽  
pp. 1495-1507 ◽  
Author(s):  
N C Collier ◽  
M J Schlesinger
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Intermediate Filament ◽  
Chicken Embryo ◽  
Dynamic State ◽  
Hsp 70 ◽  
Embryo Fibroblasts ◽  
Shock Proteins ◽  
Filament Network ◽  
Chicken Embryo Fibroblasts

Subcellular fractionation and immunofluorescence microscopy have been used to study the intracellular distributions of the major heat shock proteins, hsp 89, hsp 70, and hsp 24, in chicken embryo fibroblasts stressed by heat shock, allowed to recover and then restressed. Hsp 89 was localized primarily to the cytoplasm except during the restress when a portion of this protein concentrated in the nuclear region. Under all conditions, hsp 89 was readily extracted from cells by detergent. During stress and restress, significant amounts of hsp 70 moved to the nucleus and became resistant to detergent extraction. Some of this hsp 70 was released from the insoluble form in an ATP-dependent reaction. Hsp 24 was confined to the cytoplasm and, during restress, aggregated to detergent-insoluble perinuclear phase-dense granules. These granules dissociated during recovery and hsp 24 could be solubilized by detergent. The nuclear hsps reappeared in the cytoplasm in cells allowed to recover at normal temperatures. Sodium arsenite also induces hsps and their distributions were similar to that observed after a heat shock, except for hsp 89, which remained cytoplasmic. We also examined by immunofluorescence the cytoskeletal systems of chicken embryo fibroblasts subjected to heat shock and found no gross morphological changes in cytoplasmic microfilaments or microtubules. However, the intermediate filament network was very sensitive and collapsed around the nucleus very shortly after a heat shock. The normal intermediate filament morphology reformed when cells were allowed to recover from the stress. Inclusion of actinomycin D during the heat shock--a condition that prevents synthesis of the hsps--did not affect the intermediate filament collapse, but recovery of the normal morphology did not occur. We suggest that an hsp(s) may aid in the formation of the intermediate filament network after stress.

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.

scholarly journals A novel function for the MAP kinase SMA-5 in intestinal tube stability

2016 ◽  
Vol 27 (24) ◽  
pp. 3855-3868 ◽  
Author(s):  
Florian Geisler ◽  
Harald Gerhardus ◽  
Katrin Carberry ◽  
Wayne Davis ◽  
Erik Jorgensen ◽  
...  
Keyword(s):  
Map Kinase ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Catalytic Domain ◽  
Intestinal Tube ◽  
Reduced Body ◽  
Terminal Web ◽  
And Function ◽  
Filament Network

Intermediate filaments are major cytoskeletal components whose assembly into complex networks and isotype-specific functions are still largely unknown. Caenorhabditis elegans provides an excellent model system to study intermediate filament organization and function in vivo. Its intestinal intermediate filaments localize exclusively to the endotube, a circumferential sheet just below the actin-based terminal web. A genetic screen for defects in the organization of intermediate filaments identified a mutation in the catalytic domain of the MAP kinase 7 orthologue sma-5(kc1). In sma-5(kc1) mutants, pockets of lumen penetrate the cytoplasm of the intestinal cells. These membrane hernias increase over time without affecting epithelial integrity and polarity. A more pronounced phenotype was observed in the deletion allele sma-5( n678) and in intestine-specific sma-5(RNAi). Besides reduced body length, an increased time of development, reduced brood size, and reduced life span were observed in the mutants, indicating compromised food uptake. Ultrastructural analyses revealed that the luminal pockets include the subapical cytoskeleton and coincide with local thinning and gaps in the endotube that are often enlarged in other regions. Increased intermediate filament phosphorylation was detected by two-dimensional immunoblotting, suggesting that loss of SMA-5 function leads to reduced intestinal tube stability due to altered intermediate filament network phosphorylation.

Sign in / Sign up

Export Citation Format

Share Document