Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Summary

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

Abstract

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Introduction

Image-based analysis of fixed cells is commonly used to assess the cell population level changes in response to various perturbations. When combined with cell synchronization, followed by the collection and imaging of serial time points, such approaches can be used to suggest a cellular sequence of events. Nevertheless, fixed cell imaging is limited in that temporal relationships are implied for a population and not demonstrated at the level of individual cells. In this way, while fixed cell imaging and analysis is sufficient to observe robust phenotypes and steady-state changes, the ability to detect transient changes over time and changes that impact only a subpopul....

Protocol

1. Generation of hTERT-RPE-1 cells stably expressing RFP-Histone 2B (RFP-H2B) and α-tubulin-EGFP (tub-EGFP)

NOTE: All steps follow aseptic techniques and take place in a biosafety level II+ (BSL2+) safety cabinet.

1. Generate retrovirus carrying the genes of interest (α-tubulin-EGFP and RFP-H2B) by the transfection of 293T cells with the appropriate lentiviral plasmids according to the manufacturer's instructions of the lipid-based transfection delivery system.
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Discussion

The temporal resolution provided by the time-lapse imaging allows for the visualization and assessment of sequential cellular events within single cells. Approaches that make use of cellular synchronization followed by the collection and fixation of cells at sequential time points are limited in that comparisons are ultimately made between populations of cells. In contexts where the cellular response to perturbations may be non-uniform, or where the process being visualized is dynamic, live cell time-lapse imaging is bet.......

Materials

Name	Company	Catalog Number	Comments
0.05% Trypsin	Gibo-Life sciences	25-510	A serine protease used to release adherent cells from culture dishes
15ml centrifuge tubes	Olympus Plastics	28-101
20x CFI Plan Fluor objective	Nikon		For use in Live-cell imaging to visualize both bright field and fluorescence
293T Cells	ATCC	CRL-3216	For use in retroviral transfection; used in step 1.1
a-tubulin-EGFP	Addgene	various numbers	Expression vector for alpha tubulin fused to a green fluorescent protein tag; for use in the visualization of tubulin in live-cell imaging: commercially available through addgene and other vendors
Alisertib	Selleckchem	S1133	Small molecule inhibitor of the mitotic kinase Aurora A. Stock concentration is prepared at 10mM in DMSO, and used at a final concentration of 100nM.
Blasticidin	Invitrogen	A11139-03	Antibiotic selection agent; used to select for a-tub-EGFP expressing cells
C02	Airgas		For use in cell culture and live cell imaging
Chroma ET-DS Red (TRITC/Cy3)	Chroma	49005	Single band filter set; excitation wavelength 545nm with 25nm bandwidth and emission at 605nm wavelength with 70nm bandwidth; for visualization of H2B-RFP
Chroma ET-EGFP (FITC/Cy2)	Chroma	49002	Single band filter set; excitation wavelength 470nm with 40nm bandwidth and emission at 525nm wavelength with 50nm bandwidth; for visualization of GFP-tubulin
disposable glass Pastuer pipets, sterilized	Fisher Scientific	13-678-6A	For use in aspirating cells
Dulbecco’s Modified Eagle Medium (DMEM)	Gibo-Life sciences	11965-084	Cell culture medium for growth of RPE-1 and 293T cells
Fetal Bovine Serum (FBS)	Gibo-Life sciences	10438-026	Cell culture medium supplement
Lipofectamine 3000 and p3000	Invitrogen	L3000-015	Lipid based transfection reagent for transfection of plasmids; used in 1.1.4
Multi well Tissue Culture dishes	Corning	various	for use in cell culture, transfection/infection, and live cell imaging
Nikon Ti-E microscope	Nikon		Inverted epifluorescence microscope for use in live-cell imaging
NIS Elements HC	Nikon	Version 4.51	Image acquisition and analysis software; used in sections 3 & 4
OPTI-MEM	Gibo-Life sciences	31985-070	Reduced serum medium for cell transfection; used in step 1.1.3
Penicillin/Streptomycin	Gibo-Life sciences	15140-122	antibiotic used in cell culture medium
phosphate bufferred saline (PBS)	Caisson labs	PBP06-10X1LT	sterile saline solution for use with cell culture
pMD2.G	Addgene	12259	Lentiviral VSV-G envelope expression construct; used in step 1.1.4
Polybrene	Sigma-Aldrich	H9268	Cationic polymer used to enhane viral infection efficiency; used in step 1.1.10
psPAX	Addgene	12260	2nd generation lentiviral packaging plasmid; used in step 1.1.4
Puromycin	Invitrogen	ant-pr-1	Antibiotic selection agent; used to select for RFP-H2B expressing cells
RFP- Histone 2B (H2B)	Addgene	various numbers	Expression vector for red fluorescent protein-tagged histone 2B; for use in the visualization of chromatin in live-cell imaging: commercially available through Addgene and other vendors
RNAi Max	Invitrogen	13778-150	Lipid based transfection reagent for transfection of siRNA constructs
RPE-1 cells	ATCC	CRL-4000	Human retinal pigment epithelial cell line
Tissue culture dish 100x20mm	Corning	353003	for use in culturing adherent cells
Zyla sCMOS camera	Nikon		Camera attached to the micrscope, used for capturing images of cells