This work was supported by the Connaught Fund New Investigator Award to S.P., Canada Foundation for Innovation, NSERC Discovery Grant Program (grants RGPIN-2015-05114 and RGPIN-2020-05881), University of Manchester and University of Toronto Joint Research Fund, and University of Toronto XSeed Program.

1. Cell Seeding
NOTE: The described cell spreading protocol was performed using mouse embryonic fibroblasts (MEFs) expressing PH-Akt-GFP (a fluorescent marker for PIP3/PI(3,4)P2). This cell line was generated by genomically integrating an expression construct for PH-Akt-GFP (Addgene #21218) by CRISPR-mediated gene editing. However, other fluorescent markers that are expressed transiently or integrated in the genome can also be used in this assay. For optimal image segmentat...

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The described cell spreading assay allows for the continuous tracking of morphological changes (e.g., cell size and shape) and cell edge movements (i.e., protrusion speed and retraction frequency), which are features missing in most cell spreading protocols19,24. While commonly used end-point cell spreading assays allow for the determination of cell spreading speed, these assays fail to resolve the temporal dynamics of cell edge movements. The l...

Lamellipodial protrusions are prominent cytoskeletal structures formed at the front of a migrating cell. In lamellipodia, polymerization of actin with the aid of the Arp2/3 complex and formins&#160;creates a fast-growing branched actin meshwork that pushes against the plasma membrane1,2. The pushing force generated by the actin meshwork physically propels the cell forward1,3,4,5. Depletion of the Arp2/3 complex or disruption of signaling pathways essential for lamellipodial protrusions often impair cell migration6, 7. Although migration of lamellipodia-deficient cells has also been reported8,9, the importance of lamellipodia in cell migration is evident as depletion of this protrusive structure perturbs the cell&#39;s ability to move through&#160;complex biological microenvironments6,10.
A major hindrance to understanding the regulation of lamellipodia in migrating cells is the natural variability in lamellipodial protrusion kinetics, size, and shape11,12,13,14. Furthermore, recent studies have demonstrated that lamellipodia exhibit complex protrusive behaviors, including fluctuating, periodic, and accelerating protrusions14,15. Compared to the highly variable lamellipodia of migrating cells6,16, lamellipodia formed during cell spreading are more uniform12. Since the protrusive activity of spreading and migrating cells is driven by identical macromolecular assemblies, which include a branched actin network, contractile actomyosin bundles, and integrin-based cell-matrix adhesions17,18, spreading cells have been widely used as a model for investigating the regulation of lamellipodia dynamics.
Cell spreading is a dynamic mechanochemical process where a cell in suspension first adheres to a substrate through integrin-based adhesions17,19,20 and then spreads by extending actin-based protrusions21,22,23. During the spreading phase, lamellipodia emanating from the cell body protrude isotropically and persistently with little to no retraction or stalling12. The most commonly used cell spreading protocols are endpoints assays, where spreading cells are fixed at various times after plating19,24. These assays, although quick and simple, are limited in their diagnostic power to detect changes in the dynamic features of lamellipodia. To determine the molecular mechanisms that control lamellipodia dynamics, the Sheetz group pioneered the use of quantitative analysis of live spreading cells and uncovered many fundamental properties of cell edge protrusions11,12,22. These studies have demonstrated that the live-cell spreading assay is a robust and powerful technique in the toolbox of a cell biology laboratory. Despite that, a detailed protocol and open-source computational tool for a live-cell spreading assay are currently unavailable for the cell biology community. To this end, our protocol outlines the procedures of imaging live spreading cells and provides an automated image analysis tool. To validate this method, we used Arp2/3 inhibition as an experimental treatment and showed that inhibiting the function of the Arp2/3 complex did not arrest cell spreading but caused a significant reduction in cell protrusion speed, as well as the stability of cell edge protrusions, giving rise to jagged cell edges. These data demonstrate that the combination of live-cell imaging and automated image analysis is a useful tool for analyzing cell edge dynamics and identifying molecular components that regulate lamellipodia.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin (0.05%), 0.53 mM EDTA</td>
			<td>Wisent Bioproducts</td>
			<td>325-042-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>10.0 cm Petri Dish, Polystyrene, TC Treated, Vented</td>
			<td>Starstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL High Clarity PP Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Sterile</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Well Chamlide CMS for 22 mm x 22 mm Coverslip</td>
			<td>Quorum Technologies</td>
			<td>CM-S22-1</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm TC-treated Easy-Grip Style Cell Culture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube, Transparent, Plug Seal</td>
			<td>Nest</td>
			<td>602002</td>
			<td></td>
		</tr>
		<tr>
			<td>6.0 cm Cell Culture Dishes Treated for Increased Cell Attachment, Sterile</td>
			<td>VWR</td>
			<td>10861-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Arp2/3 Complex Inhibitor I, CK-666</td>
			<td>Millipore Sigma</td>
			<td>182515</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera, Prime 95B-25MM</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide, Sterile</td>
			<td>BioShop</td>
			<td>DMS666</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, 1x, 4.5 g/L Glucose, with L-Glutamine, Sodium Pyruvate and Phenol Red</td>
			<td>Wisent Bioproducts</td>
			<td>319-005 CL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES, No Phenol Red</td>
			<td>Gibco</td>
			<td>11039021</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS, 1X</td>
			<td>Wisent Bioproducts</td>
			<td>311-425 CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Wisent Bioproducts</td>
			<td>080-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji Software</td>
			<td>ImageJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Plasma Fibronectin Purified Protein 1 mg</td>
			<td>Millipore Sigma</td>
			<td>FC010</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil</td>
			<td>Cargille</td>
			<td>16241</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine Solution (200 mM)</td>
			<td>Wisent Bioproducts</td>
			<td>609-065-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover Glasses, Square, No. 11/2 22 x 22 mm</td>
			<td>VWR</td>
			<td>CA48366-227-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Body, Eclipse Ti2-E</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Objective, CFI Plan Apo Lambda 60X Oil</td>
			<td>Nikon</td>
			<td>MRD01605</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning Disk, Crest Light V2</td>
			<td>CrestOptics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spyder</td>
			<td>Anaconda</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stage top incubator</td>
			<td>Tokai Hit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Statistics Software, Prism</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers, Style 2</td>
			<td>Electron Microscopy Sciences</td>
			<td>78326-42</td>
			<td></td>
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quantitative analysis of cell edge dynamics during cell spreading

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out shape. Following the cell-substrate attachment, the cell forms a thin sheet of lamellipodia emanating from the cell body. In the lamellipodia, globular actin (G-actin) monomers polymerize into a dense filamentous actin (F-actin) meshwork that pushes against the plasma membrane, thereby providing the mechanical forces required for the cell to spread. Notably, the molecular players that control the actin polymerization in lamellipodia are essential for many other cellular processes, such as cell migration and endocytosis. 
Since spreading cells form continuous lamellipodia that span the entire cell periphery and persistently expand outward, cell spreading assays have become an efficient tool to assess the kinetics of lamellipodial protrusions. Although several technical implementations of the cell spreading assay have been developed, a detailed description of the workflow, which would include both a step-by-step protocol and computational tools for data analysis, is currently lacking. Here, we describe the experimental procedures of the cell spreading assay and present an open-source tool for quantitative and unbiased analysis of cell edge dynamics during spreading. When combined with pharmacological manipulations and/or gene-silencing techniques, this protocol is amenable to a large-scale screen of molecular players regulating lamellipodial protrusions.

The above protocol describes the experimental procedures for the live-cell imaging of spreading cells and a computational tool for the quantitative analysis of cell spreading dynamics. The computational tool can be used in a low- or high-throughput format to identify the molecular players regulating the actin polymerization machinery at the cell leading edge.
The schematic representation of the experimental procedures is depicted in Figure 1. The cell spreading as...

Watch this Scientific Journal Video about Quantitative Analysis of Cell Edge Dynamics during Cell Spreading at JoVE.com

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

In this protocol, we present the experimental procedures of a cell spreading assay that is based on live-cell microscopy. We provide an open-source computational tool for the unbiased segmentation of fluorescently labeled cells and quantitative analysis of lamellipodia dynamics during cell spreading.

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

The protocol described in this video will define the experimental procedures required for the life-size imaging of spreading cells and the use of a computational tool that quantifies cells spreading dynamics in an unbiased and automated fashion. The cell spreading asset allows for the continuous tracking of cell edge movement and morphological changes during cell spreading, which is a feature that is missing in most existing cell spreading assays. As well, this protocol implements the use of automated computer processing and analysis, providing information on cell circularity, area and protrusion retraction cycles of spreading cells.Such automated processing reduces bias in data analysis and provides a robust method of analyzing cell spreading dynamics for a large number of cells. When combined with drug treatments or gene science and techniques, this protocol is amenable to large-scale speeds of molecular players regulating cell protrusions. For the given protocol, The cells used are mouse embryonic fibroblasts that genetically encode PH-Akt-GFP, which allows for the fluorescent tagging of the plasma membrane.Prior to the beginning of the cell spreading assay, culture a dish of cells to 90%confluency. Once the cells have attained the proper confluency, flame a 22 by 22 millimeter cover slip and place it into a 35 millimeter cell culture dish. Coat the cover slip with fibronectin that has been diluted in PBS to a concentration of 2.5 micrograms per milliliter.Place the dish with the cover slip into a 37 degree incubator for one hour. After one hour aspirate the fibronectin making sure not to touch the cover slip with the pipette tip. Wash the dish with PBS by gently pipetting around the cover slip two to three times.For cell seeding, begin by aspirating the cell culture media from the dish of cells. Afterwards, wash the dish with warm PBS. Add 650 microliters of trypsin EDTA to the dish and tilt the dish to evenly distribute the enzyme.Place the dish with the trypsin into the incubator for one minute. After incubation you will first need to add 10 milliliters of cell culture media into a centrifuge tube. Next, quickly add another 10 milliliters of media into the dish in order to quench the trypsin.To dilute the cells that will be seeded onto the cover slip, pipette one milliliter of the dish's contents into the centrifuge tube. From the tube, pipette approximately 500 to 1, 000 microliters of diluted cells into the dish with the cover slip. Ensure that the cover slip is at a 10%confluency or 50, 000 cells per milliliter and adjust the volume of diluted cells as needed.The purpose of these cells is to establish a plane of focus during image acquisition. With the remaining cells in the dish, passage one fifth of the cells into one small dish per treatment. These will be the cells that will be analyzed for spreading dynamics.Place the passage dishes and the cover slip dish into an incubator for eight to 24 hours. To test the importance of Arp2/3 for cell spreading, first add five milliliters of cell culture media into a control and a treatment centrifuge tube. Next add 20 milliliters of dmem that is lacking phenol red into each of two larger centrifuge tubes.Pipette either the drug CK-666 which is an inhibitor of the Arp2/3 complex or the controlled treatment such as DMSO into the small and large tubes. To begin pharmacological treatment of the cells, remove the passage dishes that were incubating overnight. Aspirate the cell culture media from all of the dishes and wash the dishes with warm PBS.Take the small tubes containing either CK-666 or control supplemented media and add the contents of the relevant tube into each of the passage dishes. Label each of the dishes with the correct drug treatment and then place the dishes back into the incubator for an additional hour After one hour incubation, aspirate the drug supplemented media from each of the dishes. Next, add warm PBS to all of the dishes in order to thoroughly remove all of the remaining phenol red media.Add 230 microliters of trypsin EDTA and place the dishes into an incubator for one minute. Remove the trypsinized dishes from the incubator and proceed to add five milliliters of the drug supplemented dmem that is lacking phenol red into a tube designated as tube B.Then add another five milliliters of the same media into the relevant dish to quench the trypsin. Pipette the media up and down several times in order to detach all of the cells from the dish.Transfer all of the contents of the dish into another tube that has already been designated as tube A.In order to prepare a cell dilution that is appropriate for imaging, transfer one milliliter of cells from tube A into tube B.Repeat all of the dilution steps for each treatment. Place all of the tubes into the incubator for 45 minutes to allow cells to recover from trypsinization. Ensure that all parts of a cell magnetic chamber that can accommodate a 22 by 22 millimeter cover slip have been thoroughly cleaned before use.Remove the dish with the cover slip from the incubator. Aspirate the cell culture media and wash the cover slip with warm PBS. Remove the cover slip using a pair of forceps and gently lay the cover slip onto the bottom plate of the magnetic chamber.Next pick up the silicone gasket and place it on top of the cover slip. Attach the main body of the magnetic chamber onto the bottom plate. Add one milliliter of the dmem lacking phenol red corresponding to the relevant treatment to the magnetic chamber.Take a lint-free tissue and carefully dab the enclosure between the main body and the bottom plate in order to check for any leaks. To complete the magnetic chamber, lower the transparent cover onto the main body. Lastly wipe the bottom of the cover slip with a tissue sprayed with water and another sprayed with 70%ethanol.Preheat the stage top incubator of a confocal microscope to 37 degrees. Place the magnetic chamber on top of the objective. Prior to acquiring spreading dynamics, set focus to the already polarized cells in the green fluorescent channel.This ensures that the spreading cells will be in focus once they attach to the cover slip. Remove the transparent cover of the magnetic chamber and pipette 500 microliters from tube B that was removed from the incubator. Place the transparent cover back on top.To identify cells ideal for cell spreading analysis search for green halos which represent cells that have yet to attach to the cover slip or are in the earliest stages of attachment. Acquire images and save the files. After having completed the image acquisition of cell spreading, begin by running a compatible Python IDE such as Spyder.For computational analysis of cell spreading dynamics, locate and open the cell spreading GUI file provided in the relevant script packages. Press the run button which will open the analysis GUI panel in the background. The GUI houses all of the settings needed for analysis.To analyze cell area and circularity, input the acquisition file and the necessary settings in the cell spread area tab. Adjustments will have to be made depending on cell type and image acquisition parameters. After pressing run, the script will create a cell circularity and area versus time plot for all spreading cells.For kymograph data, press the kymograph generator and analysis tab. This analysis requires input files to be cropped single cell image stacks. After inserting all of the settings and pressing run, the script will create multiple kymographs for the given cell.On the left are representative cells from the DMSO and CK-666 treatments, automatically segmented using the provided script. As opposed to the isotropic and highly circular morphology demonstrated by control cells, Arp2/3 inhibited cells exhibit decreased circularity. Additionally, the automatically generated kymographs provide temporal resolution that is critical to understanding the dynamics of protrusions.Control cells protrude persistently with little to no retractions. In contrast, Arp2/3 inhibition disrupts the stability of protrusions as indicated by the increase in retraction events throughout spreading. This videos should provide you with the understanding of how to properly seed cells, perform pharmacological treatments and prepare imaging and computational tools necessary for the quantification of cell spreading dynamics.This protocol can be further combined with cytoskeleton fluorescent imaging and migration assays to identify the molecular players governing cell protrusions. Thank you for watching and good luck with your experiments.

quantitative-analysis-of-cell-edge-dynamics-during-cell-spreading

Research

JoVE Journal

Biology

5.2K visualizações.  University of Toronto. O protocolo descrito neste vídeo definirá os procedimentos experimentais necessários para a imagem em tamanho real das células disseminadas e o uso de uma ferramenta computacional que quantifica as células espalhando dinâmicas de forma imparcial e automatizada. O ativo de disseminação de células permite o rastreamento contínuo do movimento da borda celular e alterações morfológicas durante a disseminação celular, que é uma característica que está faltando na maioria dos ensa...