Immunofluorescent localization of protein synthesis components in mouse embryo fibroblasts

Gavrilova, L. P.; Rutkevitch, N.M.; Gelfand, Vladimir I.; Motuz, L.P.; Stahl, Joachim; Bommer, Ulrich‐Axel; Bielka, H

doi:10.1016/0309-1651(87)90134-2

Cited by 31 publications

(26 citation statements)

References 16 publications

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“…1C) and LC-MS/MS (Table III), EF-1␣ (spot 38) was an abundant protein in the tubulin-binding protein fraction. Several other elongation and initiation factors were also identified, some of which have been shown to bind to MTs and other cytoskeletal structures in previous studies (Table III) (12,(43)(44)(45)(46). These included EF-2 (spot 4) and subunits of the eukaryotic translation initiation factors eIF3 and eIF4 (spots 7,18,24,27,50,54,70).…”

Section: Resultsmentioning

confidence: 78%

Large-scale Identification of Tubulin-binding Proteins Provides Insight on Subcellular Trafficking, Metabolic Channeling, and Signaling in Plant Cells

Chuong

Good

Taylor

et al. 2004

Molecular & Cellular Proteomics

115

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Microtubules play an essential role in the growth and development of plants and are known to be involved in regulating many cellular processes ranging from translation to signaling. In this article, we describe the proteomic characterization of Arabidopsis tubulin-binding proteins that were purified using tubulin affinity chromatography. Microtubule co-sedimentation assays indicated that most, if not all, of the proteins in the tubulin-binding protein fraction possessed microtubule-binding activity. Two-dimensional gel electrophoresis of the tubulin-binding protein fraction was performed, and 86 protein spots were excised and analyzed for protein identification. A total of 122 proteins were identified with high confidence using LC-MS/MS. These proteins were grouped into six categories based on their predicted functions: microtubule-associated proteins, translation factors, RNA-binding proteins, signaling proteins, metabolic enzymes, and proteins with other functions. Almost one-half of the proteins identified in this fraction were related to proteins that have previously been reported to interact with microtubules. This study represents the first large-scale proteomic identification of eukaryotic cytoskeleton-binding proteins, and provides insight on subcellular trafficking, metabolic channeling, and signaling in plant cells. Molecular & Cellular Proteomics 3:970 -983, 2004.The cytoskeleton is the single most important structure that contributes to the highly ordered organization of the eukaryotic cell. It provides a framework for cell division and the trafficking of organelles and macromolecules, and also serves to regulate important cellular processes such as signaling, translation, and metabolism. The cytoskeleton plays a key role in a number of plant-specific processes, such as assisting in the formation of the cell plate, regulating cell-to-cell movement, and influencing the direction of cell elongation (1). A role for the microtubule (MT) 1 component of the cytoskeleton in many of these processes has been demonstrated, and a number of MT-binding proteins that are responsible for regulating these events have been identified.Plant MTs are assembled into four distinct arrays during the cell cycle (2). Three of these arrays-the interphase cortical array, the pre-prophase band, and the phragmoplast-have no counterpart in animal cells. The cortical MT array has been linked to the regulation of cellulose microfibril deposition and, hence, a role in cell expansion, while the pre-prophase band and the phragmoplast have important roles in the positioning and synthesis of the new cell plate in dividing cells. The fourth array, the spindle, has an evolutionarily conserved role in the segregation of chromosomes during cell division. The organization and dynamics of MTs in these arrays depend on the activity of various MT-associated proteins (MAPs). Several plant MAPs have been identified, including the 65-kDa MAPs, MAP 190, and MOR1 (3). These proteins are important in cross-bridging MTs, linking MTs with actin filamen...

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Section: Resultsmentioning

confidence: 78%

Large-scale Identification of Tubulin-binding Proteins Provides Insight on Subcellular Trafficking, Metabolic Channeling, and Signaling in Plant Cells

Chuong

Good

Taylor

et al. 2004

Molecular & Cellular Proteomics

115

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“…Fifteen percent to 30% of cellular mRNAs and polyribosomes are thought to associate with the cytoskeleton, and immunohistologic and biochemical approaches suggest that translation initiation and elongation factors in certain cell types follow a microtubule pattern (33). Examples of proteins that regulate translation and that associate with microtubules include eIF-2, eIF3, eIF4A, eIF-5 (34)(35)(36), the cap-binding protein eIF4E (37), eEF1a, and eEF1-2 (35). Using molecular beacons specific for HIF-1a mRNA, we observed microtubule association of HIF mRNA, which was disrupted following treatment with microtubule-depolymerizing Figure 4.…”

Section: Discussionmentioning

confidence: 99%

Both Microtubule-Stabilizing and Microtubule-Destabilizing Drugs Inhibit Hypoxia-Inducible Factor-1α Accumulation and Activity by Disrupting Microtubule Function

2005

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We have recently identified a mechanistic link between disruption of the microtubule cytoskeleton and inhibition of tumor angiogenesis via the hypoxia-inducible factor-1 (HIF-1) pathway. Based on this model, we hypothesized that other microtubule-targeting drugs may have a similar effect on HIF-1A. To test that hypothesis, we studied the effects of different clinically relevant microtubule-disrupting agents, including taxotere, epothilone B, discodermolide, vincristine, 2-methoxyestradiol, and colchicine. In all cases, HIF-1A protein, but not mRNA, was down-regulated in a drug dose-dependent manner. In addition, HIF-1A transcriptional activity was also inhibited by all drugs tested. To further examine whether these effects were dependent on microtubule network disruption, we tested the ability of epothilone B to inhibit HIF-1A protein in the human ovarian cancer cell line 1A9 and its B-tubulin mutant epothilone-resistant subclone 1A9/A8. Our data showed that epothilone B treatment down-regulated HIF-1A protein in the parental 1A9 cells but had no effect in the resistant 1A9/A8 cells. These observations were confirmed by confocal microscopy, which showed impaired nuclear accumulation of HIF-1A in parental 1A9 cells at epothilone B concentrations that induced extensive microtubule stabilization. In contrast, epothilone B treatment had no effect on either microtubules or HIF-1A nuclear accumulation in the resistant 1A9/A8 cells. Furthermore, epothilone B inhibited HIF-1 transcriptional activity in 1A9 cells, as evidenced by a hypoxia response element-luciferase reporter assay, but had no effect on HIF-1 activity in the resistant 1A9/A8 cells. These data directly link B-tubulin drug binding with HIF-1A protein inhibition. Our results further provide a strong rationale for testing taxanes and epothilones in clinical trials targeting HIF-1 in cancer patients. (Cancer Res 2005; 65(19): 9021-8)

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“…Evidence suggests that protein synthesis in mammalian cells occurs in a "channeled pathway," in which many of the components of the synthetic machinery are spatially restricted, such that individual components are directly transferred to the translational complex without dissociating into the soluble phase of the cytoplasm (Negrutskii et al, 1994). In accordance with these findings, a number of proteins that are involved in translational regulation, including the cap-binding protein (Zumbe et al, 1982), translation initiation factors eIF-2, eIF3, eIF4A, and eIF-5 (Howe and Hershey, 1984;Gavrilova et al, 1987;Heuijerjans et al, 1989), and elongation factors eEF1␣ (Yang et al, 1990) and eEF1-2 (Gavrilova et al, 1987) have been found associated with the cytoskeleton. Other proteins involved in posttranscriptional regulation such as poly A ϩ -binding protein (Greenberg, 1980) and seryl-tRNA synthetase (Miseta et al, 1991) can be found UV crosslinked to the mRNA in vivo.…”

Section: Discussionmentioning

confidence: 94%

A General RNA-Binding Protein Complex That Includes the Cytoskeleton-associated Protein MAP 1A

Defranco

Chicurel

Potter

1998

MBoC

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Association of mRNA with the cytoskeleton represents a fundamental aspect of RNA physiology likely involved in mRNA transport, anchoring, translation, and turnover. We report the initial characterization of a protein complex that binds RNA in a sequence-independent but size-dependent manner in vitro. The complex includes a ∼160-kDa protein that is bound directly to mRNA and that appears to be either identical or highly related to a ∼1600-kDa protein that binds directly to mRNA in vivo. In addition, the microtubule-associated protein, MAP 1A, a cytoskeletal associated protein is a component of this complex. We suggest that the general attachment of mRNA to the cytoskeleton may be mediated, in part, through the formation of this ribonucleoprotein complex.

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Immunofluorescent localization of protein synthesis components in mouse embryo fibroblasts

Cited by 31 publications

References 16 publications

Large-scale Identification of Tubulin-binding Proteins Provides Insight on Subcellular Trafficking, Metabolic Channeling, and Signaling in Plant Cells

Large-scale Identification of Tubulin-binding Proteins Provides Insight on Subcellular Trafficking, Metabolic Channeling, and Signaling in Plant Cells

Both Microtubule-Stabilizing and Microtubule-Destabilizing Drugs Inhibit Hypoxia-Inducible Factor-1α Accumulation and Activity by Disrupting Microtubule Function

A General RNA-Binding Protein Complex That Includes the Cytoskeleton-associated Protein MAP 1A

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