scholarly journals NuMA is required for the organization of microtubules into aster-like mitotic arrays.

1995 ◽  
Vol 131 (3) ◽  
pp. 693-708 ◽  
Author(s):  
T Gaglio ◽  
A Saredi ◽  
D A Compton
Keyword(s):  
Mitotic Spindle ◽  
Cultured Cells ◽  
Active Role ◽  
Mitotic Apparatus ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Late Stages ◽  
Temporal Accumulation

NuMA (Nuclear protein that associates with the Mitotic Apparatus) is a 235-kD intranuclear protein that accumulates at the pericentrosomal region of the mitotic spindle in vertebrate cells. To determine if NuMA plays an active role in organizing the microtubules at the polar region of the mitotic spindle, we have developed a cell free system for the assembly of mitotic asters derived from synchronized cultured cells. Mitotic asters assembled in this extract are composed of microtubules arranged in a radial array that contain NuMA concentrated at the central core. The organization of microtubules into asters in this cell free system is dependent on NuMA because immunodepletion of NuMA from the extract results in randomly dispersed microtubules instead of organized mitotic asters, and addition of the purified recombinant NuMA protein to the NuMA-depleted extract fully reconstitutes the organization of the microtubules into mitotic asters. Furthermore, we show that NuMA is phosphorylated upon mitotic aster assembly and that NuMA is only required in the late stages of aster assembly in this cell free system consistent with the temporal accumulation of NuMA at the polar ends of the mitotic spindle in vivo. These results, in combination with the phenotype observed in vivo after the prevention of NuMA from targeting onto the mitotic spindle by antibody microinjection, suggest that NuMA plays a functional role in the organization of the microtubules of the mitotic spindle.

scholarly journals Opposing motor activities are required for the organization of the mammalian mitotic spindle pole.

1996 ◽  
Vol 135 (2) ◽  
pp. 399-414 ◽  
Author(s):  
T Gaglio ◽  
A Saredi ◽  
J B Bingham ◽  
M J Hasbani ◽  
S R Gill ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Cultured Cells ◽  
Cytoplasmic Dynein ◽  
Dominant Negative ◽  
Polar Regions ◽  
Mitotic Apparatus ◽  
Independent Manner ◽  
Free System ◽  
Cell Free System

We use both in vitro and in vivo approaches to examine the roles of Eg5 (kinesin-related protein), cytoplasmic dynein, and dynactin in the organization of the microtubules and the localization of NuMA (Nu-clear protein that associates with the Mitotic Apparatus) at the polar ends of the mammalian mitotic spindle. Perturbation of the function of Eg5 through either immunodepletion from a cell free system for assembly of mitotic asters or antibody microinjection into cultured cells leads to organized astral microtubule arrays with expanded polar regions in which the minus ends of the microtubules emanate from a ring-like structure that contains NuMA. Conversely, perturbation of the function of cytoplasmic dynein or dynactin through either specific immunodepletition from the cell free system or expression of a dominant negative subunit of dynactin in cultured cells results in the complete lack of organization of microtubules and the failure to efficiently concentrate the NuMA protein despite its association with the microtubules. Simultaneous immunodepletion of these proteins from the cell free system for mitotic aster assembly indicates that the plus end-directed activity of Eg5 antagonizes the minus end-directed activity of cytoplasmic dynein and a minus end-directed activity associated with NuMA during the organization of the microtubules into a morphologic pole. Taken together, these results demonstrate that the unique organization of the minus ends of microtubules and the localization of NuMA at the polar ends of the mammalian mitotic spindle can be accomplished in a centrosome-independent manner by the opposing activities of plus end- and minus end-directed motors.

scholarly journals Authentic Replication and Recombination of Tomato Bushy Stunt Virus RNA in a Cell-Free Extract from Yeast

2008 ◽  
Vol 82 (12) ◽  
pp. 5967-5980 ◽  
Author(s):  
Judit Pogany ◽  
Peter D. Nagy
Keyword(s):  
High Efficiency ◽  
Tomato Bushy Stunt Virus ◽  
Free System ◽  
Cell Free System ◽  
Virus Rna ◽  
A Cell ◽  
Viral Replicase ◽  
Template Specificity ◽  
Rna Complex

ABSTRACT To study the replication of Tomato bushy stunt virus (TBSV), a small tombusvirus of plants, we have developed a cell-free system based on a Saccharomyces cerevisiae extract. The cell-free system was capable of performing a complete replication cycle on added plus-stranded TBSV replicon RNA (repRNA) that led to the production of ∼30-fold-more plus-stranded progeny RNAs than the minus-stranded replication intermediate. The cell-free system also replicated the full-length TBSV genomic RNA, which resulted in production of subgenomic RNAs as well. The cell-free system showed high template specificity, since a mutated repRNA, minus-stranded repRNA, or a heterologous viral RNA could not be used as templates by the tombusvirus replicase. Similar to the in vivo situation, replication of the TBSV replicon RNA took place in a membraneous fraction, in which the viral replicase-RNA complex was RNase and protease resistant but sensitive to detergents. In addition to faithfully replicating the TBSV replicon RNA, the cell-free system was also capable of generating TBSV RNA recombinants with high efficiency. Altogether, tombusvirus replicase in the cell-free system showed features remarkably similar to those of the in vivo replicase, including carrying out a complete cycle of replication, high template specificity, and the ability to recombine efficiently.

Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters

1981 ◽  
Vol 1 (7) ◽  
pp. 635-651
Author(s):  
D C Lee ◽  
R G Roeder
Keyword(s):  
Adenovirus Type ◽  
Free System ◽  
Ribonucleic Acids ◽  
Cell Free System ◽  
Cell Extracts ◽  
A Cell ◽  
Adenovirus Type 2

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

Characterization of phospholipase D in a cell-free system of cultured cells derived from rat frontal cortex

1992 ◽  
Vol 595 (1) ◽  
pp. 12-16 ◽  
Author(s):  
Akira Nishida ◽  
Masami Shimizu ◽  
Yasunori Kanaho ◽  
Yoshinori Nozawa ◽  
Shigeto Yamawaki
Keyword(s):  
Frontal Cortex ◽  
Phospholipase D ◽  
Cultured Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Rat Frontal Cortex

Yeast cell-free system that catalyses joint-molecule formation in a Rad51p- and Rad52p-dependent fashion

2000 ◽  
Vol 347 (2) ◽  
pp. 363-368 ◽  
Author(s):  
Vijayalakshmi NAGARAJ ◽  
David NORRIS
Keyword(s):  
Homologous Recombination ◽  
Specific Activity ◽  
Yeast Cells ◽  
Active Components ◽  
Free System ◽  
Cell Free System ◽  
Molecule Formation ◽  
A Cell

One of the central reactions of homologous recombination is the invasion of a single strand of DNA into a homologous duplex to form a joint molecule. Here we describe the isolation of a cell-free system from meiotic yeast cells that catalyses joint-molecule formation in vitro. The active components in the system required ATP and homologous DNA and operated in both 0.5 and 13 mM MgCl2. When the cell-free system was prepared from rad51/rad51 and rad52/rad52 mutants and joint-molecule formation was assayed at 0.5 mM MgCl2, the specific activity decreased to 6% and 13.8% respectively of the wild-type level. However, when the same mutant extracts were premixed, joint-molecule formation increased 4-8-fold, i.e. the mutant extracts exhibited complementation in vitro. These results demonstrated that Rad51p and Rad52p were required for optimal joint-molecule formation at 0.5 mM MgCl2. Intriguingly, however, Rad51p and Rad52p seemed to be more dispensable at higher concentrations of MgCl2 (13 mM). Further purification of the responsible activity has proven problematical, but it did flow through a sizing column as a single peak (molecular mass 1.2 MDa) that was co-eluted with Rad51p and RFA, the eukaryotic single-stranded DNA-binding protein. All of these characteristics are consistent with the known properties of the reaction in vivo and suggest that the new cell-free system will be suitable for purifying enzymes involved in homologous recombination.

scholarly journals Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Tomoyuki Iwasaki ◽  
Naoe Kaneko ◽  
Yuki Ito ◽  
Hiroyuki Takeda ◽  
Tatsuya Sawasaki ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Early Onset ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Blau Syndrome ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Early Onset Sarcoidosis

Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplified luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.

scholarly journals Mitotic Spindle Poles are Organized by Structural and Motor Proteins in Addition to Centrosomes

1997 ◽  
Vol 138 (5) ◽  
pp. 1055-1066 ◽  
Author(s):  
Tirso Gaglio ◽  
Mary A. Dionne ◽  
Duane A. Compton
Keyword(s):  
Mitotic Spindle ◽  
Motor Proteins ◽  
Cultured Cells ◽  
Recent Observation ◽  
Somatic Cells ◽  
Cytoplasmic Dynein ◽  
Common Mechanism ◽  
Mitotic Apparatus ◽  
Free System ◽  
Spindle Poles

The focusing of microtubules into mitotic spindle poles in vertebrate somatic cells has been assumed to be the consequence of their nucleation from centrosomes. Contrary to this simple view, in this article we show that an antibody recognizing the light intermediate chain of cytoplasmic dynein (70.1) disrupts both the focused organization of microtubule minus ends and the localization of the nuclear mitotic apparatus protein at spindle poles when injected into cultured cells during metaphase, despite the presence of centrosomes. Examination of the effects of this dynein-specific antibody both in vitro using a cell-free system for mitotic aster assembly and in vivo after injection into cultured cells reveals that in addition to its direct effect on cytoplasmic dynein this antibody reduces the efficiency with which dynactin associates with microtubules, indicating that the antibody perturbs the cooperative binding of dynein and dynactin to microtubules during spindle/aster assembly. These results indicate that microtubule minus ends are focused into spindle poles in vertebrate somatic cells through a mechanism that involves contributions from both centrosomes and structural and microtubule motor proteins. Furthermore, these findings, together with the recent observation that cytoplasmic dynein is required for the formation and maintenance of acentrosomal spindle poles in extracts prepared from Xenopus eggs (Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Nature (Lond.). 382: 420–425) demonstrate that there is a common mechanism for focusing free microtubule minus ends in both centrosomal and acentrosomal spindles. We discuss these observations in the context of a search-capture-focus model for spindle assembly.

Recombination of Foreign (Viral) DNA with the Host Genome Studies in Vivo and in a Cell-Free System

1987 ◽  
pp. 60-72 ◽  
Author(s):  
W. Doerfler ◽  
A. Spies ◽  
R. Jessberger ◽  
U. Lichtenberg ◽  
C. Zock ◽  
...  
Keyword(s):  
Host Genome ◽  
Viral Dna ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Cytochrome P -450 synthesis in vivo and in a cell-free system from rat liver

FEBS Letters ◽  
1978 ◽  
Vol 89 (2) ◽  
pp. 337-340 ◽  
Author(s):  
Kolari S. Bhat ◽  
G. Padmanaban
Keyword(s):  
Rat Liver ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Cytochrome P 450
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