Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function

Summary

The purpose of this protocol is to fuse two different cell types to create hybrid cells. Fluorescence microscopy analysis of fused cells is used to track the cell of origin of cellular organelles. This assay can be used to explore how cellular structure and function respond to perturbation by cell fusion.

Abstract

Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion is the merger of two or more cells to form a single cell. Here we provide a protocol for cell fusion of two different cell types. Fused hybrid cells are enriched by flow cytometry-based sorting, followed by fluorescence microscopy of hybrid cell structure and function. Fluorescently tagged proteins generated by genome editing are imaged inside fused cells, allowing cellular structures to be identified based on fluorescence emission and referenced back to the cell type of origin. This robust and general method can be applied to different cell types or organelles of interest, to understand cellular structure and function across a range of fundamental biological questions.

Introduction

Homeostatic maintenance of cellular structure is critical to life. Cells have characteristic morphologies, sub-cellular organelle numbers, and internal biochemical composition. Understanding how these fundamental properties are generated and how they go awry during disease requires laboratory tools to perturb them.

Cell fusion is the merging of two or more separate cells. Cell fusion may have been critical to the emergence of eukaryotic life¹. In the human body, cell fusion is relatively rare, occurring during restricted developmental circumstances and tissue types, such as during fertilization or the formation of mu....

Protocol

1. Differential Fluorescent Cell Labelling

1. Gene tagging with CRISPR Cas9
   1. Use CRISPR Cas9 genome editing to tag rootletin (or other genes of interest) with the fluorescent proteins meGFP or mScarlet-I in human cancer cell lines.
      NOTE: Detailed protocols for genome editing are covered elsewhere^24,25,26.
2. Fluorescent dye labelling
   1. Grow Cal51 human cancer cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), L-glutamine, and 100 µg/mL penic....

Results

Appropriately labelled cells are visible during flow cytometry by fluorescence signal higher than unlabeled control cells (Figure 2A). Gates are set for sorting of double positive cells, enriching this population directly into imaging dishes for further microscopic analyses. Fused cells are detectable as distinct double fluorescently positive cells and constitute about ~1% of the population.

Discussion

We demonstrate a facile and cost-effective protocol for fusing cells and visualizing the subsequent architecture of cell hybrids with microscopy, taking approximately two days from start to finish. Critical parts of this protocol are the enrichment of fused cells by cell sorting (protocol section 3), and careful validation of fused cells by microscopy (protocol section 4). These sections ensure that fused cells are readily obtained and are bona fide heterokaryons. Concentrations and incubation times should be adhered to........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL tube	Sarstedt	62554502
37% formaldehyde solution	Sigma-Aldrich	F8875
880 Laser Scanning Confocal Airyscan Microscope	Carl Zeiss
8-well imaging dishes	Ibidi	80826
Anti-GFP alpaca GFP booster nanobody	Chromotek	gba-488
BD Influx Cell Sorter	BD Biosciences
Bovine serum albumin	Sigma-Aldrich	A7906
Cell Filters (70 μm)	Biofil	CSS010070
CellTrace Far Red	ThermoFisher Scientific	C34572
CellTrace Violet	ThermoFisher Scientific	C34571
Dulbecco's Modified Eagle Medium (DMEM), high glucose, GlutaMAX, pyruvate	ThermoFisher Scientific	31966021
Fetal Bovine Serum	Sigma-Aldrich	10270-106
FluoTag-X2 anti-mScarlet-I alpaca nanobody	NanoTag Biotechnologies	N1302-At565
L15 CO2 independent imaging medium	Sigma-Aldrich	21083027
Penicillin/streptomycin	Sigma-Aldrich	15140122
Phenol red free DMEM, high glucose	ThermoFisher Scientific	21063029
Phosphate buffered saline (1x PBS)			8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2HPO4, dH2O up to 1 L
Polyethylene Glycol Hybri-Max 1450	Sigma-Aldrich	P7181
Polypropylene tubes	BD Falcon	352063
Triton X-100	Fisher BioReagents	BP151	nonionic surfactant
Trypsin	Sigma-Aldrich	T4049
Tween 20	Fisher BioReagents	BP337	nonionic detergent