PubMed · 14821537
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1951-04-14. MITOSIS.. https://pubmed.ncbi.nlm.nih.gov/14821537/
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BACKGROUND: All eukaryotes with the exception of plants use an actomyosin ring to generate a constriction force at the site of cell division (cleavage furrow) during mitosis and meiosis. The structure and filament forming abilities located in the C-terminal or tail region of one of the main components, myosin II, are important for localising the molecule to the contractile ring (CR) during cytokinesis. However, it remains poorly understood how myosin II is recruited to the site of cell division and how this recruitment relates to myosin filament assembly. Significant conservation between species of the components involved in cytokinesis, including those of the CR, allows the use of easily genetically manipulated organisms, such as budding yeast (Saccharomyces cerevisiae), in the study of cytokinesis. Budding yeast has a single myosin II protein, named Myo1. Unlike most other class II myosins, the tail of Myo1 has an irregular coiled coil. In this report we use molecular genetics, biochemistry and live cell imaging to characterize the minimum localisation domain (MLD) of budding yeast Myo1. RESULTS: We show that the MLD is a small region in the centre of the tail of Myo1 and that it is both necessary and sufficient for localisation of Myo1 to the yeast bud neck, the pre-determined site of cell division. Hydrodynamic measurements of the MLD, purified from bacteria or yeast, show that it is likely to exist as a trimer. We also examine the importance of a small region of low coiled coil forming probability within the MLD, which we call the hinge region. Removal of the hinge region prevents contraction of the CR. Using fluorescence recovery after photobleaching (FRAP), we show that GFP-tagged MLD is slightly more dynamic than the GFP-tagged full length molecule but less dynamic than the GFP-tagged Myo1 construct lacking the hinge region. CONCLUSION: Our results define the intrinsic determinant for the localization of budding yeast myosin II and show it to be an oligomer of tentatively 3 monomers. We suggest that this is the minimum oligomeric unit (rather than the traditional myosin II dimer) that would allow specific assembly to the site of cytokinesis in a manner similar to the full length molecule. The flexible hinge region also contributes to CR structural integrity and contractility.
To meet the requirements of the EU 7th Amendment to the Cosmetics Directive, manufacturers of cosmetics products will need to ascertain the safety of ingredients using non-animal methods. Starting in 2009, in vivo genotoxicity tests for cosmetics ingredients will not be allowed. Skin is a target area of interest for many cosmetic products because of its relatively high exposure. Therefore, it would be beneficial to have a non-animal, skin-based genotoxicity assay, especially one that utilized human skin in vitro. In this paper, we describe the development of a reproducible micronucleus assay that uses EpiDerm engineered human skin constructs (MatTek Corp., Ashland, MA). We describe methods for isolating single cells from the 3D skin model and for processing the cells for microscopic analysis of micronuclei (MN). In addition, since little was known about the kinetics of the dividing keratinocytes in the EpiDerm model, we evaluated whether cytochalasin B (Cyt-B) could be used to distinguish the population of dividing cells allowing the development of a micronucleus assay in binucleated cells. We found that the frequency of binucleated cells increased both with time and with increasing concentration of Cyt-B. After a 48-h exposure, 30-50% binucleated cells were reproducibly obtained. Finally, we evaluated micronucleus induction using the model genotoxicants mitomycin C (MMC) and vinblastine sulfate (VB). The background frequency of MN is very low and reproducible in this model, and statistically significant increases in the frequency of micronucleated cells were induced by both MMC and VB. These are initial steps in developing a routine "in vivo-like" assay for chromosomal damage in human tissue. It is hoped that other investigators utilize these methods to further the understanding of this potentially valuable new non-animal method.
In this review, we discuss the recent identification of ARF-BP1 (also known as Mule, UREB1, E3(histone), LASU1, and HectH9). ARF-BP1, a HECT domain-containing E3 ubiquitin ligase, interacts with ARF and p53. Its ubiquitin ligase activity is inhibited by ARF. Inactivation of ARF-BP1 stabilised p53 and induced apoptosis. Notably, inactivation of ARF-BP1 also caused cell growth repression in p53-null cells and breast cancer cells with mutant p53. Thus, ARF-BP1 emerges as a novel therapeutic target against cancer regardless of p53 status.