title: Studying proteolysis of cyclin B at the single cell level in whole cell populations.
abstract: Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/C(Cdc20) until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths. Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects. To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B. To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium.
authors.affiliation: Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg.
authors.forename: Dominik
authors.initials: D
authors.lastname: Schnerch
authors.forename: Marie
authors.initials: M
authors.lastname: Follo
authors.forename: Julia
authors.initials: J
authors.lastname: Felthaus
authors.forename: Monika
authors.initials: M
authors.lastname: Engelhardt
authors.forename: Ralph
authors.initials: R
authors.lastname: Wäsch
mesh_terms: D001859:Bone Neoplasms / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D019926:Cyclin B / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D012516:Osteosarcoma / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D050600:SNARE Proteins / Q000737:chemistry; D059010:Single-Cell Analysis / Q000379:methods*; D008941:Spindle Apparatus / Q000737:chemistry / Q000378:metabolism
chemical_list: D019926:Cyclin B; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins