Elucidating Human Mitosis Using an Anaphase-Like Cell-Free System

Abstract

A balanced progression through mitosis and cell division is largely dependent on orderly phosphorylation and ubiquitin-mediated proteolysis of regulatory and structural proteins. These series of events ultimately secure genome stability and time-invariant cellular properties during cell proliferation. Two of the core enzymes regulating mitotic milestones in all eukaryotes are cyclin dependent kinase 1 (CDK1) with its coactivator cyclin B, and the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Discovering mechanisms and substrates for these enzymes is vital to understanding how cells move through mitosis and segregate chromosomes with high fidelity. However, the study of these enzymes has significant challenges. Purely in vitro studies discount the contributions of yet to be described regulators and misses the physiological context of cellular environment. In vivo studies are complicated by the fact that each of these enzymes, as well as many of their regulators and downstream targets, are essential. Moreover, long-term in vivo manipulations can result in cascading, indirect effects that can distort data analysis and interpretation. Many of these challenges can be circumvented using cell-free systems, which have historically played a critical role in identifying these enzymes and their contributions under quasicellular environments. Here, we describe the preparation of a newly developed human cell-free system that recapitulates an anaphase-like state of human cells. This new toolkit complements traditional cell-free systems from human cells and frog eggs and can be easily implemented in cell biology labs for direct and quantitative studies of mitotic signaling regulated by phosphorylation, APC/C-mediated proteolysis, and beyond.

Keywords: APC/C; Anaphase; Cdc20; Cdh1; Cdk1; Cell extracts; Cell-free system; Mitosis; Nondegradable cyclin B; Ubiquitin-mediated degradation.

Fig. 1

NDB cell- and cell-free systems for elucidating human mitosis. (a) NDB cell system is based on human 293-T-REx^™ cells stably expressing nondegradable (ND) mutant of Cyclin B1 under a tetracycline (Tet)/doxycycline (Dox)-regulated CMV promoter. Expression of ND-Cyclin B1 induces mitotic arrest in an anaphase-like state. The constant activity of the Cdk1–Cyclin B1 complex prevents mitotic exit and maintains a peak level of APC/C^Cdc20 activity for hours. NDB extracts are typically prepared from 20 plates of 150 mm/diameter. Recommended cell confluency for Tet-induced expression is 75–80%. Following 20–22 h treatment with Tet, cells acquire a spherical shape. This archetypal morphology indicates mitotic arrest. Cell synchronization should be further validated by standard DNA quantification. The arrested cells will be loosely attached to the surface, if at all. Thus, cells can be collected directly by gentle pipetting (without trypsinization) into a single 500 mL conical tube. Once cells are pelleted and washed, they are ready for extract preparation. In vitro translated (IVT) protein products are generated in reticulocyte lysate supplemented with radiolabeled methio-nine (³⁵S-Met). pCS2-based expression vectors and reticulocyte lysates supporting transcription from SP6 promoter are recommended. A typical reaction mix contain 10–20 μL NDB mitotic extracts and 0.5–1.5 μL IVT product. Extracts already contain energy-regeneration mix (E-mix), as well as endogenous Ubiquitin (Ub) and the E2 enzyme UbcH10. As such, extracts are highly active. Yet adding 0.5–1 μL of ×20 E. mix, recombinant Ub, and UncH10 into the reaction mix can facilitate proteolysis mediated by APC/C^Cdc20. Conversely, proteasome inhibitors (e.g., MG132), dominant negative UbcH10 (UbcH10^DN), or APC/C specific inhibitors (e.g., Emi1 or TAME) can be added for validating the potency and specificity of the assay. Assays are typically performed in a temperature range of 23–30 °C. During degradation/mobility-shift assays, 3–5 μL of the reaction mix is sampled each time point, mixed with Laemmli buffer, boiled and frozen at 80 °C. Protein sampled are resolved by SDS-PAGE. Gels are then soaked in a distain solution, heat/vacuum dried, and exposed to a Phosphorimager screen for 1 h to 1 day. Longer exposure might be helpful in case of weak signals. The potency and specificity of NDB mitotic extracts is demonstrated; Geminin, an APC/C^Cdc20 substrate, but not Tome-1, an APC/C^Cdh1 substrate, is degraded. This degradation is blocked by Emi1. Orderly phosphorylation of Tome-1 is evident in minute-scale resolution by a gradual mobility shift. The high mobility shift of Tome-1 observed after 140 min indicates for the stability and potency of this cell-free system

Fig. 2

Characterization of APC/C^Cdc20-mediated degradation in NDB mitotic extracts. (a) Time-dependent degradation of Securin (³⁵S-labeled, IVT product) in mitotic NDB extracts was assayed in three different temperatures. Extracts were supplemented with E-mix and Ub (0.5 mg/mL). The impact of adding recombinant UbcH10 or UbcH10^DN (0.5 mg/mL) on the degradation of Securin also tested at 28 °C. Degradation was assayed by SDS–PAGE and autoradiography. Quantification of the depicted raw data (left) are plotted on the right. Data are normalized to max signal at t = 0. (b) Time-dependent degradation of Securin in diluted NDB mitotic extracts are shown (28 °C). See (a) for details. NDB extracts were diluted two- or fourfold in swelling buffer

Fig. 3

Mitotic NDB cells and extracts can exit mitosis by Cdk1 inhibition. NDB cells induced by Tet are static in an anaphase-like state. These cells can, however, progress into G1 following treatment with the Cdk1 inhibitor RO-3306. Phase images and DNA distributions are shown before and after 2.5 h treatment with RO-3306. Similarly, RO-3306 induces mitotic exit in NDB extracts. The resulting G1-like extracts support the degradation of Tome-1 (APC/C^Cdh1 substrate). Moreover, the mitotic—Cdk1-dependent—mobility shift of this protein (top right panel) is no longer apparent (bottom right panels). Tome-1 degradation is G1-like NDB extracts is facilitated by UbcH10 and blocked by MG132. These results demonstrate the shift from APC/C^Cdc20- to APC/C^Cdh1-specific activity in mitotic vs. G1-like NDB extracts. Degradation of Tome-1 (³⁵S-labeled, IVT product) was assayed in 28 °C, and resolved by SDS-PAGE and autoradiography