In vitro reassembly of squid brain intermediate filaments (neurofilaments): purification by assembly-disassembly

Science ◽  
1980 ◽  
Vol 208 (4448) ◽  
pp. 1152-1155 ◽  
Author(s):  
RV Zackroff ◽  
RD Goldman
Keyword(s):  
Brain Tissue ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Total Protein ◽  
Dense Bodies ◽  
Or Organization

Intermediate filaments from squid brain tissue were reassembled in vitro and purified by two cycles of assembly and disassembly. Purified squid brain filaments contained one major polypeptide (60,000 daltons), which constituted about 70 percent of the total protein, and three minor polypeptides (74,000, 100,000, and 220,000 daltons). Squid brain intermediate filaments were reconstituted from rod-shaped protofilamentous subunits. In addition to the intermediate filaments, dense bodies which may function in intermediate filament nucleation or organization were retained through two purification cycles.

scholarly journals alpha-Internexin, a 66-kD intermediate filament-binding protein from mammalian central nervous tissues.

1985 ◽  
Vol 101 (4) ◽  
pp. 1316-1322 ◽  
Author(s):  
J S Pachter ◽  
R K Liem
Keyword(s):  
Optic Nerve ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Peptide Mapping ◽  
Exchange Chromatography ◽  
Filament Assembly ◽  
Antigen Antibody

In this paper we describe a 66-kD protein that co-purifies with intermediate filaments from rat optic nerve and spinal cord but can be separated further by ion-exchange chromatography. This protein is distinct from the 68-kD neurofilament subunit protein as judged by isoelectric focusing, immunoblotting, peptide mapping, and tests of polymerization competence. This protein is avidly recognized by the monoclonal anti-intermediate filament antigen antibody, previously demonstrated to recognize a common antigenic determinant in all five known classes of intermediate filaments. Also, when isolated this protein binds to various intermediate filament subunit proteins, which suggests an in vivo interaction with the intermediate filament cytoskeleton, and it appears to be axonally transported in the rat optic nerve. Because of this ability to bind to intermediate filaments in situ and in vitro we have named this protein alpha-internexin. A possible functional role for the protein in organizing filament assembly and distribution is discussed.

Expression of a developmentally regulated cross-linking intermediate filament-associated protein (IFAPa-400) during the replacement of vimentin for desmin in muscle cell differentiation

1991 ◽  
Vol 98 (2) ◽  
pp. 251-260 ◽  
Author(s):  
L.J. Cossette ◽  
M. Vincent
Keyword(s):  
Chick Embryo ◽  
Muscle Cell ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cell Types ◽  
Complete Disappearance ◽  
Developmentally Regulated ◽  
Complete Replacement ◽  
Embryo Cells

Myogenic and neurogenic tissues of the chick embryo transiently express IFAPa-400, a high molecular weight protein that colocalizes and is copurified with intermediate filaments. Using monoclonal antibody F51H2 to identify it, we carried out immunoelectron microscopy experiments on whole-mount chick embryo cells and showed that IFAPa-400 was localized at crossing points of intermediate filaments. Also, immunoblot experiments with F51H2, anti-vimentin and anti-desmin antibodies demonstrated the complete disappearance of IFAPa-400 in those muscle cell types that change their vimentin content for desmin during embryogenesis. During in vitro myogenesis, the expression of IFAPa-400 was shown to be concurrent with the progressive replacement of vimentin by desmin in myoblasts. When long-term myotube cultures were maintained on a fibroblast-like cell layer, we observed the complete replacement of vimentin by desmin, followed by the disappearance of IFAPa-400 from the myotubes. These results suggest that IFAPa-400 might be involved in the reorganization of the intermediate filament network during muscle differentiation.

Purification of developmentally regulated avian 400-kDa intermediate filament associated protein. Molecular interactions with intermediate filament proteins and other cytoskeleton components

1995 ◽  
Vol 73 (9-10) ◽  
pp. 651-657 ◽  
Author(s):  
Marie Duval ◽  
Xiaoying Ma ◽  
Jean-Paul Valet ◽  
Michel Vincent
Keyword(s):  
Chick Embryo ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Specific Binding ◽  
Developmentally Regulated ◽  
Intermediate Filament Proteins ◽  
In Vitro Recombination ◽  
Associated Proteins ◽  
Structural Lattice

IFAPa-400, a 400-kDa developmentally regulated protein thought to be associated with intermediate filaments, has been purified from chick embryo hearts to investigate its interaction with vimentin and other IF proteins and to identify other cellular components to which this cytoskeletal protein associates. Previous studies suggested that this protein was associated with the vimentin-containing intermediate filament lattice of myoblasts and neuroblasts before their terminal differentiation, providing these cells with a particular intermediate filament cytoskeleton that could satisfy specific mechanical requirements during their intense morphogenetic activities. Although IFAPa-400 partially reassociated with vimentin and desmin in disassembly–reassembly experiments using crude IF preparations from chick embryo hearts, in vitro recombination of purified IFAPa-400 with vimentin and desmin failed to demonstrate any direct association. When purified IFAPa-400 was used as a probe in blot overlay assays, however, specific binding to vimentin and desmin was observed, providing the first evidence of a physical association between IFAPa-400 and intermediate filament proteins. The blot overlay experiments also demonstrated that IFAPa-400 binds to two unidentified polypeptides of 19 and 32 kDa. These results are thus consistent with the hypothesis that a structural lattice requiring a vimentin–IFAPa-400 combination constitutes the intermediate filament system of myogenic and neurogenic cells.Key words: cytoskeleton, intermediate filaments, intermediate filament associated proteins, vimentin, IFAPa-400.

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 Cytoskeleton-associated plectin: in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins.

1988 ◽  
Vol 106 (3) ◽  
pp. 723-733 ◽  
Author(s):  
R Foisner ◽  
F E Leichtfried ◽  
H Herrmann ◽  
J V Small ◽  
D Lawson ◽  
...  
Keyword(s):  
Immunoelectron Microscopy ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Solid Phase ◽  
Chinese Hamster ◽  
Structural Element ◽  
Intermediate Filament Proteins ◽  
Rat Glioma ◽  
Branching Points

The association and interaction of plectin (Mr 300,000) with intermediate filaments and filament subunit proteins were studied. Immunoelectron microscopy of whole mount cytoskeletons from various cultured cell lines (rat glioma C6, mouse BALB/c 3T3, and Chinese hamster ovary) and quick-frozen, deep-etched replicas of Triton X-100-extracted rat embryo fibroblast cells revealed that plectin was primarily located at junction sites and branching points of intermediate filaments. These results were corroborated by in vitro recombination studies using vimentin and plectin purified from C6 cells. Filaments assembled from mixtures of both proteins were extensively crosslinked by oligomeric plectin structures, as demonstrated by electron microscopy of negatively stained and rotary-shadowed specimens as well as by immunoelectron microscopy; the binding of plectin structures on the surface of filaments and cross-link formation occurred without apparent periodicity. Plectin's cross-linking of reconstituted filaments was also shown by ultracentrifugation experiments. As revealed by the rotary-shadowing technique, filament-bound plectin structures were oligomeric and predominantly consisted of a central globular core region of 30-50 nm with extending filaments or filamentous loops. Solid-phase binding to proteolytically degraded vimentin fragments suggested that plectin interacts with the helical rod domain of vimentin, a highly conserved structural element of all intermediate filament proteins. Accordingly, plectin was found to bind to the glial fibrillar acidic protein, the three neurofilament polypeptides, and skin keratins. These results suggest that plectin is a cross-linker of vimentin filaments and possibly also of other intermediate filament types.

scholarly journals Site specificity in vimentin-membrane interactions: intermediate filament subunits associate with the plasma membrane via their head domains.

1985 ◽  
Vol 100 (6) ◽  
pp. 1962-1967 ◽  
Author(s):  
S D Georgatos ◽  
D C Weaver ◽  
V T Marchesi
Keyword(s):  
Plasma Membrane ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Membrane Vesicles ◽  
Polymerization Process ◽  
Acceptor Site ◽  
Site Specificity ◽  
Amino Terminal ◽  
10 Nm Filaments

Fragments of vimentin, generated by chemical or enzymatic cleavages, were analyzed for their capacity to bind to human inverted erythrocyte membrane vesicles. Only peptides comprising the amino-terminal head domain of vimentin molecules were competent in associating with the membranes. In vitro studies also demonstrated that isolated ankyrin (the major vimentin acceptor site on the membrane) binds to an oligomeric species of vimentin and prevents the formation of characteristic 10-nm filaments. These data, taken together with the observation that the NH2-terminal end of vimentin is implicated in the polymerization process (Traub, P., and C. Vorgias, J. Cell Sci., 1983, 63:43-67), imply that intermediate filaments may contact the membrane in an end-on fashion, using the exposed head domains of their terminal subunits.

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.

Comparative Study of Size, Total Protein, Fibrinogen and 5-HT Content of Human and Canine Platelet Density Subpopulations

1986 ◽  
Vol 56 (03) ◽  
pp. 288-292 ◽  
Author(s):  
Diego Mezzano ◽  
Eduardo Aranda ◽  
Arnaldo Foradori
Keyword(s):  
Comparative Study ◽  
Protein Content ◽  
Cell Density ◽  
Total Protein ◽  
Unit Volume ◽  
Low Density ◽  
Dense Bodies ◽  
Artifactual Changes ◽  
Platelet Release ◽  
The Difference

SummaryThe size, total protein, fibrinogen and 5-HT content were evaluated in density subpopulations of human and canine platelets fractionated in linear arabinogalactan gradients. The methodology was assessed to ascertain that platelet separation was by density and to discard artifactual changes and platelet release during the procedure. EDTA or PGEi increased the size of human PRP-platelets, but not of dog platelets. In humans, high density (HD) platelets were 1.26 times larger and contained 1.88 times more fibrinogen, 2.23 times more 5-HT and 1.37 times more protein than low density (LD) platelets; in dogs, these density cohorts did not differ in protein content, but LD platelets were 1.29 times larger and had 1.33 times more fibrinogen and 5-HT than HD platelets. These findings suggest that cell density is mostly dependent on the protein content per unit volume of platelets (and not on dense bodies). The differences in fibrinogen and 5-HT content between HD and LD cohorts in humans and dogs may be related to platelet age. The difference in volume between HD and LD platelets in dogs is of uncertain interpretation.

Monocytes as Carriers of Magnetic Nanoparticles for Tracking Inflammation in the Epileptic Rat Brain

2019 ◽  
Vol 16 (7) ◽  
pp. 637-644 ◽  
Author(s):  
Hadas Han ◽  
Sara Eyal ◽  
Emma Portnoy ◽  
Aniv Mann ◽  
Miriam Shmuel ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Ex Vivo ◽  
In Vivo Studies ◽  
Nanoparticle Distribution ◽  
Spontaneous Seizures ◽  
Systemic Distribution ◽  
Hippocampal Ca1 ◽  
Pilocarpine Model

Background: Inflammation is a hallmark of epileptogenic brain tissue. Previously, we have shown that inflammation in epilepsy can be delineated using systemically-injected fluorescent and magnetite- laden nanoparticles. Suggested mechanisms included distribution of free nanoparticles across a compromised blood-brain barrier or their transfer by monocytes that infiltrate the epileptic brain. Objective: In the current study, we evaluated monocytes as vehicles that deliver nanoparticles into the epileptic brain. We also assessed the effect of epilepsy on the systemic distribution of nanoparticleloaded monocytes. Methods: The in vitro uptake of 300-nm nanoparticles labeled with magnetite and BODIPY (for optical imaging) was evaluated using rat monocytes and fluorescence detection. For in vivo studies we used the rat lithium-pilocarpine model of temporal lobe epilepsy. In vivo nanoparticle distribution was evaluated using immunohistochemistry. Results: 89% of nanoparticle loading into rat monocytes was accomplished within 8 hours, enabling overnight nanoparticle loading ex vivo. The dose-normalized distribution of nanoparticle-loaded monocytes into the hippocampal CA1 and dentate gyrus of rats with spontaneous seizures was 176-fold and 380-fold higher compared to the free nanoparticles (p<0.05). Seizures were associated with greater nanoparticle accumulation within the liver and the spleen (p<0.05). Conclusion: Nanoparticle-loaded monocytes are attracted to epileptogenic brain tissue and may be used for labeling or targeting it, while significantly reducing the systemic dose of potentially toxic compounds. The effect of seizures on monocyte biodistribution should be further explored to better understand the systemic effects of epilepsy.

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