The nucleolar structure and the activity of NopA100, a nucleolin-like protein, during the cell cycle in proliferating plant cells

Abstract

For the purpose of gaining knowledge of the relationships between cell proliferation and ribosome biogenesis, as two fundamental mutually interconnected cellular processes, studies were performed on cell populations synchronized in their cell-cycle progression by treatment with hydroxyurea, followed by sampling at different times after its removal. A structural rearrangement of the nucleolus was observed throughout the interphase, along with changes in the relative amounts of different nucleolar subcomponents. A structural model of nucleolar organization was associated with each interphase period. Throughout interphase, the nucleolin-like protein, NopA100, was immunodetected in the dense fibrillar component of the nucleolus, preferentially near fibrillar centers and its levels were shown to increase from G1 to G2. A western blotting analysis of soluble nuclear protein extracts with anti-NopA100 antibody resulted in the intense labeling of a 100-kDa band, but also of a series of proteins related to it, suggesting that NopA100 undergoes a physiological process of proteolytic maturation, similar to that described for mammalian nucleolin, but not reported in other biological model systems. Physiological proteolysis of NopA100, related to cell-cycle progression, was confirmed after the nuclei extracted from synchronized cells were treated with the protease inhibitor, leupeptin, which resulted in an increase of the 100-kDa band at the expenses of the decrease of some other bands, according to the cell-cycle stages. We therefore conclude that there is a relationship between the increase in nucleolar activity, cell-cycle progression, nucleolar structure, the activity of NopA100, and the proteolysis of this nucleolin-like protein.

Germination of Arabidopsis Seed in Space and in Simulated Microgravity: Alterations in Root Cell Growth and Proliferation

“…This parameter was used to estimate the proliferation rate of the root meristematic cells, according to previous publications in which the accuracy of this estimation was established (Beemster and Baskin 1998). Increased cell proliferation was accompanied by a decrease in nucleolar activity, which was assessed according to the demonstrated correlation of this physiological parameter with several morphometric parameters of the nucleolus (nucleolar size, proportion of granular component), as well as with the amounts of the nucleolar protein, nucleolin (Medina et al 2000;González-Camacho and Medina 2006). Consequently, the production of ribosomes, the protein factories of the cell, was reduced, as was cell growth (Bernstein and Baserga 2004;Baserga 2007).…”

A bona fide La protein is required for embryogenesis in Arabidopsis thaliana

“…The plant nucleoli have the particularity as compared to other eukaryotes, to display a central entity named nucleolar cavity that appears as a clear space when observed by electron microscopy (75,76). The precise function of the nucleolar cavity is not well defined but amongst other factors small nuclear and nucleolar RNAs were shown to localize in this space (76–78).…”

“…We also observed that, in a small fraction of cells, a significant pool of AtLa1 localizes to the nucleolar cavity. The nucleolar cavity is a highly dynamic entity, devoid of transcription, whose presence and structure is dependent upon cell cycle progression (75,76). Since we did not utilize a synchronous cell population to perform subcellular localization experiments, not every observed protoplast will contain a nucleolar cavity, and since the structure and probably content of this entity varies at least according to the cell cycle, it is possible that AtLa1 does not accumulate in this sub-nucleolar compartment at all times.…”

Characterization of AtNUC-L1 Reveals a Central Role of Nucleolin in Nucleolus Organization and Silencing of AtNUC-L2 Gene in Arabidopsis

“…Immunofluorescence was performed using 4-and 8-d-old (WT and Atnuc-L1, respectively) A. thaliana seedlings following the procedures for immunocytological and structural analysis described previously (Gonzalez-Camacho and Medina, 2005). Treated seedlings were incubated with ␣-NUC1 diluted 1:1000 or with ␣-NUC2 diluted 1:250 for 16 h at 37°C and then with fluorochrome A-488 -conjugated goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO) diluted 1:100, for 3 h at 37°C.…”