पूरे सेल आबादी में एकल कोशिका के स्तर पर Cyclin बी के proteolysis अध्ययन
Summary
संक्रमण Anaphase Anaphase जटिल (APC / सी) पर निर्भर ubiquitination और cyclin बी के बाद यहाँ विनाश को बढ़ावा देने, हम एक प्रणाली है, जो नाड़ी – पीछा लेबलिंग के बाद स्थापित, पूरे सेल आबादी में cyclin बी proteolysis निगरानी की अनुमति देता है और के माध्यम से शुरू हो रहा है मेटाफ़ेज़ mitotic जांच की चौकी से हस्तक्षेप का पता लगाने की सुविधा है.
Abstract
Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. 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 spindle1. 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/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
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 5.
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 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (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 7 (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 6 (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 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.
Protocol
Discussion
हम यहाँ एक जीवित कोशिका इमेजिंग आधारित दृष्टिकोण cyclin बी proteolysis और गुणसूत्र संरेखण के साथ – साथ निगरानी की सुविधा मौजूद है. इस दृष्टिकोण की अनुमति देता है mitotic में नियंत्रण के अध्ययन एकल कोशिका के स्तर पर सेल …
Disclosures
The authors have nothing to disclose.
Acknowledgements
एस टेलर को pLPCX Histone H2 GFP प्लाज्मिड प्रदान करने के लिए आभारी हैं. हम निरंतर समर्थन के लिए आर Mertelsmann धन्यवाद. यह काम ड्यूश Forschungsgemeinschaft द्वारा समर्थित किया गया.
Materials
| Name of product | Company | Catalogue number | Comments (optional) |
| Reporter cell line | generated in-house as described 6 | clone 11 reporter cells (U2Os-based cyclin B-SNAP expressing cells) |
|
| Retroviral cyclin B-SNAP expression vector | generated in-house as described 6 | pLNCX2-cyclin B mut5-SNAP | |
| Phenolred-free DMEM | Gibco | 21063-029 | Supplementation with FCS, sodium pyruvate, penicillin/strepto-mycin required |
| SNAP-Cell TMR-Star | New England Biolabs | S9105S | Stock solution 400 μM in DMSO |
| Special Opstics Plate, 96 well | Costar | 3720 | |
| μ-Slide 8 well, ibiTreat | Ibidi | 80826 | |
| Microscopy unit | Olympus | IX-81 inverse microscope with climate chamber | |
| Objective | Olympus | UPLSAPO 20x objective (N.A. 0.75) | |
| Acquisition software | Olympus | Scan^R Acquisition software (v.2.2.09) | |
| Analysis software | Olympus | Scan^R Analysis software (v.1.2.0.6) |
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