Live Cell Imaging of Chromosome Segregation During Mitosis

Summary

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Abstract

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56γ regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

Introduction

In the following protocol, we describe a convenient method to visualize the chromosomal segregation and mitotic progression during cell cycle in mouse embryonic fibroblasts using Histone 2B-GFP, BacMam 2.0 labeling and live cell imaging.

Cell cycle control checkpoints monitor chromosome segregation and play an important role in the maintenance of the genetic integrity of the cell ^1,2,3. Accumulation of mis-segregated chromosomes can lead to aneuploidy, which is a hallmark of most solid tumors ⁴. Hence, detection of lagging c....

Protocol

All the experiments conducted in these studies were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at the Food and Drug Administration (FDA) research facility.

1. Isolation and Culture of Mouse Embryonic Fibroblasts (MEFs)

1. Isolate mouse embryonic fibroblasts (MEFs) from a PP2A-B56γ- mouse strain and wild type littermates by standard protocol ^7,8,9.
2. ExpandMEFs for 3 passages, freeze and store until needed for experiments ⁸.

Results

MEFs from wild type and PP2A-B56γ- mice were seeded in a 2-well chamber cover glass and allowed to attach. On day 2, MEFs were synchronized using 0.1% FBS for 24 h. On day 3, MEFs in media with 200 ng/mL nocodazole and 30 PPC CL-HB regentwere incubated for 18 h at 37^{°C and 5% CO}₂. On day 4, the cells were imaged using a spinning disk confocal microscope system (Figure 1). Live cell imaging was used to visualize the fate of individual cel.......

Discussion

Cell cycle control checkpoints that ensure accurate chromosome segregation prevent aneuploidy and cell transformation ^1,2,3. In the present study, we found that inactivation of PP2A-B56γ resulted in a weakened spindle assembly checkpoint. Live cell imaging allowed us to observe chromosomal mis-segregation during mitosis in PP2A-B56γ MEFs treated with nocodazole ⁹.

Thi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM/F12	Gibco	11320082
L-Glutamine	Gibco	25030081
MEM Non-Essential amino acids solution (100X)	Gibco	11140050
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140122
Dulbecco’s Phosphate Buffered saline	Gibco	14190144
Trypsin-EDTA	Gibco	25300054
Fetal bovine serum	Atlanta Biologicals	S11150
DMSO	EMD Millipore	MX 1458-6
Cryogenic vial storage boxes	Fisherbrand	10-500-28
Cryogenic vials	Corning/costar	431416
T75 flasks	Cellstar	658175
2 well chambered cover glass	Nunc	155380PK
Cellometer Vision Cell Profiler	Nexcelom Bioscience LLC	Cellometer Vision Trio
Nocodazole	Sigma	M1404
CellLight Histone 2B-GFP, BacMam 2.0	Thermo Fisher Scientific Inc.	C10594
Zeiss Cell Observer Spinning Disk Confocal Microscope system	Carl Zeiss Microscopy	Zeiss Cell Observer SD
Water Bath	Thermoscientific	280 series
Incubator	Sanyo commercial solutions	MCO-18AIC (UV)
Class II Biological safety cabinet	The Baker Company	SterilGard
Axiovision software	Zeiss	Ver.4.8.2
ImageJ software	National Institute of Health	Ver. 1.51r