Cytoskeleton and Small G Proteins (Progress in Molecular and Subcellular Biology)
L-glutamine, salt pyruvate, non-essential amino acids, streptomycin, phosphate-buffered saline PBS and nuclear fluorochrome Hoechst were obtained from Sigma-Aldrich St. Primary antibodies against RhoA monoclonal mouse; sc; dilution, and fibrillarin mouse monoclonal; sc; dilution, were obtained from Santa Cruz Biotechnology, Inc.
Actinomycin D was additionally obtained from Sigma-Aldrich. Shanghai, China. Goat anti-mouse immunoglobulin Ig G fluorescein isothiocyanate-conjugated FITC; ; dilution, , and goat anti-mouse IgG horseradish peroxidase HRP -conjugated ; dilution, , secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc.
A stock solution of actinomycin D 2. It was diluted at least 2,fold and freshly prepared prior to addition to the cells. For the reversibility assay, HEp-2 cells grown on cover slips in a 6-well plate Genetimes Biological Mall, Shanghai, China were treated with actinomycin D. Following treatment, cover slips were washed 3 times 1 min each wash in prewarmed PBS, followed by 1 brief wash with prewarmed fresh cell culture medium without actinomycin D.
Subsequent to treatment with actinomycin D at the concentrations of 0. HEp-2 cells grown on cover slips were fixed with freshly prepared paraformaldehyde Nantong Jiangtian Chemical Co. Cells were washed three times with PBS 10 min each wash subsequent to each incubation. Cells were washed with cold PBS and transferred to a 1. The supernatant was collected as whole cell protein extract. GAPDH was used as a loading control. Following the incubation of the membranes with the anti-IgG HRP-conjugated secondary antibody for 1 h at room temperature, ECL reagents were used to indicate the positive bands on the membrane, according to the manufacturer's protocol.
A comparison of the amount of total RhoA protein following treatment with varying concentrations of actinomycin D was evaluated by one-way analysis of variance using SPSS Confocal laser scanning microscopy revealed the nuclear localization of RhoA with predominant accumulation in the cell nucleolus in HEp-2 cells with no drug treatment Fig. Corresponding differential interference contrast pictures revealed large nucleoli in untreated HEp-2 cells Fig.
Accompanied by a decrease in nucleolar staining, a bright nucleoplasmic speckle staining of RhoA was visualized in the cells Fig. However, the segregated nucleoli or nucleolus-like bodies were observed in the cells treated with actinomycin D Fig. A Indirect immunofluorescence revealed prominent nucleolar RhoA in untreated cells. B Corresponding cells were revealed in differential interference contrast pictures. Redistribution of nucleolar RhoA to nucleoplasmic speckles was observed in the cells treated with actinomycin D at the concentrations of C 0.
Arrows represent the segregated nucleoli or nucleolus-like bodies. RhoA, ras homolog family member A. Intracellular localization of two abundant nucleolar proteins fibrillarin and B23 was additionally examined by indirect immunofluorescence Fig.
Nucleolar protein B23 is well-known to relocalize following treatment with actinomycin D in numerous cell types, including HEp-2 cells 21 — The staining for B23 was concentrated in the nucleoli of untreated HEp-2 cells Fig. This redistribution of B23 may act as a marker for effective treatment by actinomycin D.
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Another abundant nucleolar protein, fibrillarin, demonstrated exclusively nucleolar localization in untreated cells Fig. Consistent with the previous finding, a redistribution of fibrillarin to the nucleoplasmic small entities was induced by actinomycin D Fig. Actinomycin D altered the subcellular location of nucleolar proteins B23 and fibrillarin in HEp-2 cells. Cells showed nucleolar localization of B23 A without treatment and B with 0.
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B23 translocated from the nucleoli to the nucleoplasm in a diffuse staining pattern. C and D Corresponding cell nuclei were shown by nuclear staining with Hoechst Cells demonstrated nucleolar localization of fibrillarin E without treatment and F with 0.
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Fibrillarin redistributed to small nucleoplasmic entities. G and H Corresponding cell nuclei were shown by nuclear staining with Hoechst B23, nucleolar phosphoprotein B Following treatment with actinomycin D for 4 h, HEp-2 cells were washed with PBS and lysed under denaturing conditions.
The protein amounts from whole-cell lysate were compared through immunoblotting analysis. Regardless of the various concentrations of actinomycin D, immunoblotting revealed a single band exclusively at 21 kDa for all untreated and actinomycin D-treated groups Fig. Therefore, the actinomycin D treatment did not affect the net protein amount of RhoA, whilst it did affect the cellular distribution of the RhoA protein.
Immunoblotting of RhoA protein amount in whole HEp-2 cell lysate. Total RhoA protein was detected as a single band at 21 kDa in untreated cells and cells treated with actinomycin D for 4 h at the concentration of 0. Glyceraldehydephosphate dehydrogense was used as a loading control. To investigate the persistence and reversibility of the actinomycin D-induced nuclear redistribution of RhoA, HEp-2 cells were initially treated with actinomycin D, followed by an additional cultivation in fresh culture medium without actinomycin D for at least 24 h. When HEp-2 cells were incubated with 0.
Removal of the drug from the culture medium allowed the reaccumulation of RhoA in the cell nucleoli 24 h later Fig. Localization of ras homolog family member A in HEp-2 cells following the removal of actinomycin D. A Cells treated with actinomycin D at a concentration of 0.
Heterotrimeric G Proteins and the Regulation of Microtubule Assembly
B Cells treated with actinomycin D at a concentration of 0. Over the past few decades, the understanding of nucleolar function has changed markedly with the concept of the plurifunctional nucleolus 9. Proteomic studies have suggested that the cell nucleolus may be involved in a wide range of cellular processes independent of ribosome biogenesis Extensive evidence indicates that the nucleolus affects the response to cellular stress, and regulates the cell cycle and cell growth Perturbations to the nucleolus have been reported in a wide range of cellular diseases, from autoimmunity to cancer An important aspect regarding nucleolus structure and function are the morphological variations that exist between normal somatic and neoplastic or malignant cells For over a century, an increase in the size and number of cell nucleoli has been utilized as a marker of aggressive tumors 31 , Contemporary studies have inferred that nucleoli may have a broad role in malignant transformation.
Specifically, the extra-ribosomal functions of the nucleolus position the organelle as a central integrator of cellular proliferation and stress signaling and are emerging as important mechanisms for modulating how oncogenes and tumor suppressors operate in normal and malignant cells Research has demonstrated that RhoA serves a key role in governing extra- and intracellular signaling transduction, in addition to being involved in numerous biological processes, including tumorigenesis RhoA, as the most extensively investigated member of the Rho protein family, acts as a molecular switch in cells, regulating signal transduction from cell surface receptors to intracellular target molecules, and is involved in a number of biological processes, including cell morphology, motility and tumor progression Without destroying the cells, the present study demonstrated a redistribution of nucleolar RhoA between the nucleoli and the nucleoplasm with a speckled staining pattern following the treatment of HEp-2 cells with actinomycin D.
Furthermore, immunoblotting revealed a single band exclusively at 21 kDa, indicating that an actinomycin D-induced decrease in nucleolar RhoA was not accompanied by any alteration in RhoA protein content, biochemical modification or cleavage of RhoA.
These results are concordant with previous findings regarding the nucelolar protein fibrillarin following treatment with actinomycin D. The present results demonstrating that the nucleolar protein B23 translocated to the nucleoplasm, along with redistribution of nucleolar fibrillarin to the nucleoplasm following treatment with actinomycin D, are also consistent with previous observations made in various types of cells 24 , At low concentrations 0.
The reaccumulation of RhoA occurred between the nucleoplasm and the nucleoli following removal of the lower concentration 0. However, due to the action of actinomycin D on RNA polymerases II and III at increased concentrations, even subsequent to an extended period of drug removal, the displacement of RhoA was irreversible upon treatment of cells with increased concentrations of actinomycin D, which led to severe inhibition of RNA synthesis.
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Although the exact location of RhoA in respect to the nucleolar component remains to be elucidated, considering the similar nucleolar localization of RhoA and transcription sites, the observation of actinomycin D-induced redistribution of nucleolar RhoA to the nucleoplasm revealed direct evidence that the residency of RhoA in the nucleolus was dependent on active transcription. Increasing evidence emerged that these cellular proteins may be dynamic, crossing through the cytoplasm, nucleoplasm and nucleolus to perform their roles, and a number of these proteins may remain to be elucidated 16 , 18 , In the current era of translational medicine, the newly recognized roles of the nucleolus raise the possibility that cancer drug discovery and emerging chemotherapy may identify a foundation in the nucleolus or nucleolar proteins to a greater degree than had been anticipated Knowledge of the location, function and duration of the residency of proteins in the nucleolus is vital in identifying additional drug targets in the nucleolus.
In conclusion, the results of the present study provided in situ evidence that actinomycin D induced a redistribution of nucleolar RhoA to the nucleoplasm in human carcinoma HEp-2 cells. In addition, the results indicated that the residency of RhoA in the cell nucleolus is associated with active RNA synthesis. To the best of our knowledge, these results provide the first information regarding nucleolar RhoA in association with nucleolar function.
Additional investigations on the nuclear or nucleolar functioning of RhoA are underway and may provide novel considerations for cancer therapy. Misteli T: Protein dynamics: Implications for nuclear architecture and gene expression. Trends Cell Biol. J Histochem Cytochem. The recruitment of the BBSome to the base of the cilium depends first on its localization to the centrioles. It is still unclear why mutations in CEP can give such a diversity of phenotypes.
Depletion of Cep results in the accumulation of PCM1 at the centrosomes. There are two proposed models for the transport of ciliary proteins inside the ciliary membrane: lateral diffusion and targeted vesicle transport. RAB11 controls vesicle exit from the recycling endosomes, 34 and RAB8A is involved in vesicular trafficking between the trans-golgi network and the plasma membrane.
Since Rabin8 is not found in the cilium or the CSs, it seems that this interaction with BBS1 must occur in the basal body. Rabin8 forms a key bridge between the vesicular membranes and the BBSome, forming a direct interaction only with BBS1.