Checkpoint controls ensure the completion of cell cycle events with high fidelity in the correct order. Here we show the existence of a novel checkpoint that ensures coupling of cell wall synthesis and mitosis. In response to a defect in cell wall synthesis, S. cerevisiae cells arrest the cell-cycle before spindle pole body separation. This arrest results from the regulation of the M-phase cyclin Clb2p at the transcriptional level through the transcription factor Fkh2p. Components of the dynactin complex are required to achieve the G2 arrest whilst keeping cells highly viable. Thus, the dynactin complex has a function in a checkpoint that monitors cell wall synthesis.
BackgroundIn breast cancer cells, the metastatic cell state is strongly correlated to epithelial-to-mesenchymal transition (EMT) and the CD44+/CD24- stem cell phenotype. However, the MCF-7 cell line, which has a luminal epithelial-like phenotype and lacks a CD44+/CD24- subpopulation, has rare cell populations with higher Matrigel invasive ability. Thus, what are the potentially important differences between invasive and non-invasive breast cancer cells, and are the differences related to EMT or CD44/CD24 expression?MethodsThroughout the sequential selection process using Matrigel, we obtained MCF-7-14 cells of opposite migratory and invasive capabilities from MCF-7 cells. Comparative analysis of epithelial and mesenchymal marker expression was performed between parental MCF-7, selected MCF-7-14, and aggressive mesenchymal MDA-MB-231 cells. Furthermore, using microarray expression profiles of these cells, we selected differentially expressed genes for their invasive potential, and performed pathway and network analysis to identify a set of interesting genes, which were evaluated by RT-PCR, flow cytometry or function-blocking antibody treatment.ResultsMCF-7-14 cells had enhanced migratory and invasive ability compared with MCF-7 cells. Although MCF-7-14 cells, similar to MCF-7 cells, expressed E-cadherin but neither vimentin nor fibronectin, β-catenin was expressed not only on the cell membrane but also in the nucleus. Furthermore, using gene expression profiles of MCF-7, MCF-7-14 and MDA-MB-231 cells, we demonstrated that MCF-7-14 cells have alterations in signaling pathways regulating cell migration and identified a set of genes (PIK3R1, SOCS2, BMP7, CD44 and CD24). Interestingly, MCF-7-14 and its invasive clone CL6 cells displayed increased CD44 expression and downregulated CD24 expression compared with MCF-7 cells. Anti-CD44 antibody treatment significantly decreased cell migration and invasion in both MCF-7-14 and MCF-7-14 CL6 cells as well as MDA-MB-231 cells.ConclusionsMCF-7-14 cells are a novel model for breast cancer metastasis without requiring constitutive EMT and are categorized as a "metastable phenotype", which can be distinguished from both epithelial and mesenchymal cells. The alterations and characteristics of MCF-7-14 cells, especially nuclear β-catenin and CD44 upregulation, may characterize invasive cell populations in breast cancer.
Background: The cell wall has an important role in maintaining cell shape. In the budding yeast Saccharomyces cerevisiae, the major filamentous component of the cell wall responsible for its rigidity is 1,3‐β‐glucan and is synthesized by 1,3‐β‐glucan synthase (GS), localized on the plasma membrane. Results: Observations of green fluorescent protein (GFP)‐conjugated Fks1p, a catalytic subunit of GS, revealed that it is co‐localized with cortical actin patches and moves on the cell surface at the sites of cell wall remodelling. Mutants with impaired actin patch movement show immobility of Fks1p‐GFP spots, indicating that actin patch motility is required for the movement of Fks1p. Cells with immobilized Fks1p exhibit defective cell wall structure and function. The cell wall thickness of the mutants becomes irregular, eventually leading to cell lysis. Conclusion: We propose that GS movement is necessary for proper cell wall remodelling.
Saccharomyces cerevisiae is a multifunctional molecular switch involved in establishment of cell morphogenesis. We systematically characterized isolated temperature-sensitive mutations in the RHO1 gene and identified two groups of rho1 mutations (rho1A and rho1B) possessing distinct functional defects. Biochemical and cytological analyses demonstrated that mutant cells of the rho1A and rho1B groups have defects in activation of the Rho1p effectors Pkc1p kinase and 1,3--glucan synthase, respectively. Heteroallelic diploid strains with rho1A and rho1B mutations were able to grow even at the restrictive temperature of the corresponding homoallelic diploid strains, showing intragenic complementation. The ability to activate both of the essential Rho1p effector proteins was restored in the heteroallelic diploid. Thus, each of the complementing rho1 mutation groups abolishes a distinct function of Rho1p, activation of Pkc1p kinase or 1,3--glucan synthase activity.After establishment of cell polarity, morphogenesis of plant and fungal cells is determined by organization of the intracellular cytoskeleton and construction of the extracellular cell wall. A Rho-type small GTP-binding protein (Rho1p) in the budding yeast Saccharomyces cerevisiae has been shown to play a pivotal role in cell morphogenesis by regulating its effector proteins. Rho1p binds and activates Fks1p and Fks2p, two closely related catalytic subunits of 1,3--glucan synthase (GS), 1 thereby directly controlling cell wall synthesis (1, 2). Rho1p also binds and activates Pkc1p, a yeast homolog of mammalian protein kinase C. Through the mitogen-activated protein kinase (MAPK) cascade, Pkc1p regulates organization of the actin cytoskeleton and transcription of several genes involved in cell wall integrity (3-6). Other Rho1p-interacting proteins include Bni1p, Skn7p, and Sec3p (7-10). Gene disruption analyses revealed that among the five Rho1p effector proteins, Fks1/2p and Pkc1p are the most important in yeast cell growth. ⌬fks1⌬fks2 and ⌬pkc1 are both lethal in complete medium (11, 12), whereas ⌬sec3 shows slow growth in synthetic medium (13), and ⌬bni1 and ⌬skn7 display normal growth (7, 15). Thus, Rho1p controls cell morphogenesis by regulating the activities of two essential effector proteins important for cell wall synthesis and actin cytoskeleton organization.Several conditional lethal mutations (high temperature-sensitive mutations) in the RHO1 gene (rho1-2, rho1-3, rho1-4, rho1-5) have been isolated in our laboratory and characterized for elucidation of Rho1p function. Biochemical analyses of the rho1 mutants greatly contributed to the understanding of the essential pathways downstream of Rho1p (2, 5, 6). However, the rho1 mutants did not always exhibit a single unique phenotype. Helliwell et al. (6) reported that actin morphologies differ among the rho1 mutants: rho1-3 and rho1-4 display normal polarized actin patches, whereas rho1-2 and rho1-5 possess delocalized actin patches. In this study, we investigated what kind of phenotypic differences ...
Using probes obtained by PCR amplification, we have isolated two cognate rice cDNAs (cdc2Os-1 and cdc2Os-2) encoding structural homologues of the cdc2+/CDC28 (cdc2) protein kinase from a cDNA library prepared from cultured rice cells. Comparison of the deduced amino acid sequences of cdc2Os-1 and cdc2Os-2 showed that they are 83% identical. They are 62% identical to CDC28 of Saccharomyces cerevisiae and much more similar to the yeast and mammalian p34cdc2 kinases than to rice R2, a cdc2-related kinase isolated previously by screening the same rice cDNA library with a different oligonucleotide probe. Southern blot analysis indicated that the three rice clones (cdc2Os-1, cdc2Os-2 and R2) are derived from distinct genes and are each found in a single copy per rice haploid genome. RNA blot analysis revealed that these genes are expressed in proliferating rice cells and in young rice seedlings. cdc2Os-1 could complement a temperature-sensitive yeast mutant of cdc28. However, despite the similarity in structure, both cdc2Os-2 and R2 were unable to complement the same mutant. Thus, the present results demonstrate the presence of structurally related, but functionally distinct cognates of the cdc2 cell cycle kinase in rice.
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