This work was funded by a Wellcome Trust Henry Wellcome Fellowship to R.M. (https://wellcome.ac.uk/grant number 100090/12/Z). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Ashok Venkitaraman and Paul French for critical advice and guidance on the project. We thank Chiara Cossetti and Gabriela Grondys-Kotarba in the Cambridge Institute for Medical Research Flow Cytometry facility for excellent support. We thank Liam Cassiday, Thomas Miller, and Gianmarco Contino for proofreading the manuscript.

1. Differential Fluorescent Cell Labelling
<ol>
	<li>Gene tagging with CRISPR Cas9
	<ol>
		<li>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 elsewhere24,25,26.</li>
	</ol>
	</li>
	<li>Fluorescent dye labelling
	<ol>
		<li>Grow Cal51 human c...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+cellular+fusion+using+optically+trapped+plasmonic+nano-heaters.">Controlled cellular fusion using optically trapped plasmonic nano-heaters.</a> Optical Trapping and Optical Micromanipulation XIII. 9922, 992211 (2016).</li><li>Kao, K. N., Michayluk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+high-frequency+intergeneric+fusion+of+plant+protoplasts.">A method for high-frequency intergeneric fusion of plant protoplasts.</a> Planta. 115 (4), 355-367 (1974).</li><li>Ahkong, Q. F., Fisher, D., Tampion, W., Lucy, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cell+fusion.">Mechanisms of cell fusion.</a> Nature. 253 (5488), 194-195 (1975).</li><li>Mahen, R., Venkitaraman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+centrosome+assembly.">Pattern formation in centrosome assembly.</a> Current Opinion in Cell Biology. 24 (1), 14-23 (2012).</li><li>Mahen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+centrosomal+roots+disentangle+to+allow+interphase+centriole+independence.">Stable centrosomal roots disentangle to allow interphase centriole independence.</a> PLoS Biology. 16 (4), e2003998 (2018).</li><li>Mahen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+assessment+of+fluorescent+transgene+methods+for+quantitative+imaging+in+human+cells.">Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.</a> Molecular Biology of the Cell. 25 (22), 3610-3618 (2014).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature Protocols. 8 (11), 2281-2308 (2013).</li><li>Yang, J., Shen, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylene+glycol-mediated+cell+fusion.">Polyethylene glycol-mediated cell fusion.</a> Methods in Molecular Biology. 325, 59-66 (2006).</li><li>Huang, L., Chen, Y., Huang, W., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+pairing+and+polyethylene+glycol+(PEG)-mediated+cell+fusion+using+two-step+centrifugation-assisted+single-cell+trapping+(CAScT).">Cell pairing and polyethylene glycol (PEG)-mediated cell fusion using two-step centrifugation-assisted single-cell trapping (CAScT).</a> Lab on a Chip. 18 (7), 1113-1120 (2018).</li><li>Feliciano, D., Nixon-Abell, J., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggered+Cell-Cell+Fusion+Assay+for+Cytoplasmic+and+Organelle+Intermixing+Studies.">Triggered Cell-Cell Fusion Assay for Cytoplasmic and Organelle Intermixing Studies.</a> Current Protocols in Cell Biology. 81 (1), e61 (2018).</li><li>Vaughan, V. 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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....

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 muscle, bone and the placenta2. This protocol describes the induction of cell-cell fusion in tissue culture cell lines with differentially fluorescently labelled organelles, as a tool to understand the mechanisms controlling cell structure and function.
In vitro induced cell-cell fusion is central to the production of monoclonal antibodies3, an important tool for biological research and disease treatment. Cell fusion has also been used to ask many different fundamental cell biological questions about cell cycle dominance4, aneuploidy5,6, cellular reprogramming7,8, the repair of damaged neurons9, viral proliferation10, apoptosis11, tumorigenesis12, cytoskeletal dynamics13, and membrane fusion14,15. Laboratory based methods to induce cell-cell fusion16,17,18,19 induce lipid membrane coalescence through the physical merging of two bilayers into one. Cell fusion can be induced by electricity18, viral based methods17, thermoplasmonic heating20, transgene expression19, and chemicals including polyethylene glycol (PEG)16,21,22.
Centrosomes are microtubule organizing centers controlling cellular shape, motility, polarization, and division23. Centrosomal roots are fibrous structures extending from centrosomes containing the protein rootletin24 (encoded by the gene CROCC). We recently used cell-cell fusion to understand how centrosome position and number varies inside heterokaryons relative to parental cells24. The rationale behind the use of this method is to track the cell of origin of roots within a heterokaryon after fusion of differentially fluorescently tagged parental cells, and thus to image organelle fusion and fission. The fluorescently tagged proteins rootletin-meGFP or rootletin-mScarlet-I are created by genome editing in separate cell lines which are then fused by PEG-mediated cell fusion. We describe the use of cell dyes (Table of Materials) to identify fused cells by flow cytometry and subsequent fluorescence microscopy identification of centrosome cell of origin and morphology (Figure 1). This approach is a robust and unique method to study how major changes in cellular state including organelle number impinge upon cell homeostasis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 ml tube</td>
			<td>Sarstedt</td>
			<td>62554502</td>
			<td></td>
		</tr>
		<tr>
			<td>37% formaldehyde solution</td>
			<td>Sigma-Aldrich</td>
			<td>F8875</td>
			<td></td>
		</tr>
		<tr>
			<td>880 Laser Scanning Confocal Airyscan Microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8-well imaging dishes</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP alpaca GFP booster nanobody</td>
			<td>Chromotek</td>
			<td>gba-488</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Influx Cell Sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Filters (70um)</td>
			<td>Biofil</td>
			<td>CSS010070</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace Far Red</td>
			<td>ThermoFisher Scientific</td>
			<td>C34572</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace Violet</td>
			<td>ThermoFisher Scientific</td>
			<td>C34571</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM), high glucose, GlutaMAX, pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>31966021</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoTag-X2 anti-mScarlet-I alpaca nanobody</td>
			<td>NanoTag Biotechnologies</td>
			<td>N1302-At565</td>
			<td></td>
		</tr>
		<tr>
			<td>L15 CO2 independent imaging medium</td>
			<td>Sigma-Aldrich</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red free DMEM, high glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>21063029</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (1 x PBS)</td>
			<td></td>
			<td></td>
			<td>8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2HPO4, dH2O up to 1L</td>
		</tr>
		<tr>
			<td>Polyethylene Glycol Hybri-Max 1450</td>
			<td>Sigma-Aldrich</td>
			<td>P7181</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tubes</td>
			<td>BD Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher BioReagents</td>
			<td>BP151</td>
			<td>nonionic surfactant</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher BioReagents</td>
			<td>BP337</td>
			<td>nonionic detergent</td>
		</tr>
	</tbody>
</table>

cell-cell fusion of genome edited cell lines for perturbation of cellular structure and function

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.

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.
<p class="jove_content" fo:keep-together.within...

Watch this Scientific Journal Video about Cell-cell Fusion of Genome Edited Cell Lines for Perturbation of Cellular Structure and Function at JoVE.com

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

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.

Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion ...

This method allows cellular structure and function to be perturbed through the fusion of two different cell types to address questions such as how do cells respond to changes in organelle number. By imaging fluorescently tagged proteins within fused cells, cellular structures can be referenced back to their cell type of origin to study the mechanisms of organelle homeostasis and function. For fluorescent dye labeling, see the human cancer cell populations of interest such that each culture will have expanded to at least six times 10 to the sixth cells at a 70 to 90%confluency by the next morning.On the day of labeling, wash the rootletin eGFP and rootletin-mScarlet cells two times with 10 milliliters of PBS per wash. Dilute Violet cell dye to 500 nanomolar in PBS and Far Red cell dye to 200 nanomolar in PBS. Label the rootletin eGFP cells with the diluted Violet cell dye and the rootletin-mScarlet cells with the diluted Far Red cell dye for one minute at room temperature.At the end of the incubations, stop the reactions with 10 milliliters of DMEM for five minutes. Wash both labeled cell cultures and one unlabeled parental cell culture with 10 milliliters of fresh PBS before incubating all of the cultures with one milliliter of prewarmed trypsin. At the end of the incubation, transfer the detached cell solutions to individual 15 milliliter conical tubes for centrifugation and gently resuspend the dye labeled pellets in one milliliter of PBS and the unlabeled cells in one milliliter of DMEM.Then return all of the cell samples to the cell culture incubator. For cell fusion experiment, mix 500 microliters of Violet dye labeled cells with 500 microliters of Far Red dye labeled cells in a new 15 milliliter tube and sediment the remaining unmixed labeled cells by centrifugation. Resuspend the pellets in one milliliter of fresh DMEM and place the cells back into the incubator.Collect the mixed cells by centrifugation and add 700 microliters of 50%1450 PEG in a drop-wise manner to the pellet over a period of 30 seconds. After 3.5 minutes at room temperature, add 10 milliliters of serum-free DMEM drop-wise over a period of 30 seconds and place the tubes into the incubator for 10 minutes. At the end of the incubation, sediment the mixed cells by centrifugation and gently resuspend the pellet in one milliliter of complete medium.To enrich for the fused cell population by FACS, gently filter all of the cells into individual FACS tubes through a 70 micrometer strainer and create forward scatter versus side scatter and doublet discrimination plots in the cytometer software. Run the unlabeled cells to record their fluorescence intensity and create gates to identify the fused cells. Next, run the single positive Violet dye labeled cells to confirm that there is no spillover of Violet signal into the Far Red channel and the single positive Far Red dye labeled cells to confirm that there is no Far Red signal in the Violet channel.Then briefly run the fusion sample to confirm that the fused cells are visible with these gates. When the sorting parameters have been set, align the sorting stream centrally into an eight-well imaging dish containing 100 microliters of growth medium and sort the double positive fused cells directly into the dish. When all of the cells have been sorted, immediately place the dish into the cell culture incubator for two to 16 hours.For immunofluorescent staining, first replace the cell culture supernatant with 200 microliters of 4%paraformaldehyde for a 15-minute incubation at room temperature. At the end of the fixation, wash the cells three times with 200 microliters of PBS per wash before permeabilizing the cells in 200 microliters of 0.1%nonionic surfactant for 10 minutes at room temperature. Next, block the nonspecific binding with 200 microliters of 3%bovine serum albumin in PBS for 30 minutes at room temperature followed by labeling with the antibodies of interest in 150 microliters of staining buffer.After one hour at room temperature, wash the cells two times in 300 microliters of PBS for five minutes per wash. After the last wash, replace the wash with 200 microliters of fresh PBS. To acquire images of the fused cells, use an appropriate fluorescence microscope capable of four-color imaging and collect three-dimensional images.Appropriately labeled cells are visible during flow cytometry by fluorescent signal at a higher level than unlabeled control cells. The gates can be set for sorting to enrich for the double positive cell population directly into imaging dishes for further microscopic analysis. Fusion induces a major rearrangement of the cellular architecture through the mixing of two cells into one.Heterokaryons are identified as cells containing both fluorescent dye signals mixed inside a single cell without intervening plasma membranes. Additionally, two nuclei may also be visible in fused cells by Brightfield imaging. The cell structure and function may be further investigated through the merging of cells containing meGFP and mScarlet tagged proteins.Fusion results in a doubling of the centrosome number inside heterokaryons visible as at least four pericentriolar material foci when the centrosome pericentriolar component NEDD1 is fluorescently tagged. The fusion of cells with endogenously fluorescently tagged rootletin allows the cell of origin of each centrosome to be identified in a heterokaryon. This protocol demonstrates how to fuse differentially labeled cell lines and how to image their organelles to understand their structure and function.This robust protocol is both relatively easy and relatively inexpensive to perform compared to similar methods. This technique can be used to ask a range of questions such as how is organelle fusion regulated and how the cells respond to changes in subcellular organelle number.

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Research

JoVE Journal

Developmental Biology

8.9K visualizações.  Imperial College London. Este método permite que a estrutura e a função celular sejam perturbadas através da fusão de dois tipos diferentes de células para abordar questões como como as células respondem às mudanças no número de organela. Por meio de proteínas fluorescentes marcadas fluorescentes dentro de células fundidas, as estruturas celulares podem ser referenciadas de volta ao seu tipo de origem celular para estudar os mecanismos da organela homeostase e função. Para rotular corantes fluorescente...