Name: measuring cell-edge protrusion dynamics during spreading using live-cell microscopy
Uploaded: 2021-11-01T13:00:00
Description: Watch this Scientific Journal Video about Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy at JoVE.com

This work was supported by grants NIH MH115939, NS112121, NS105640, and R56MH122449-01A1 (to Anthony J. Koleske) and from the Israel Science Foundation (grants number 1462/17 and 2142/21) (to Hava Gil-Henn).

All methods described in this protocol have been approved by the institutional Animal Care and Use Committee (IACUC) of Bar-Ilan University.
NOTE: A step-by-step graphical depiction of the procedure described in this section appears in Figure 1.
1. Cell culture
NOTE: The cells used in the protocol are mouse embryonic fibroblasts (MEFs) that were generated from E11.5-13.5 embryos of wild-type C5...

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Cell-edge protrusion dynamics, comprised of protrusions, retractions, and ruffles, is both a prerequisite and a potential rate-limiting event in cell motility. Here we describe a fast and simple method for measuring the dynamics of cell-edge protrusions during spreading. This method enables short-time imaging, generates a significant amount of data, does not require fluorescent labeling of cells or expensive fluorescent microscopy equipment, and could be used as a preliminary method for testing cytoskeletal dynamics invo...

Cell migration is a critical process that controls the development and function of all living organisms. Cell migration occurs in both physiological conditions, such as embryogenesis, wound healing, and immune response, and in pathological conditions, such as cancer metastasis and autoimmune disease. Despite differences in cell types that take part in different migratory events, all cell motility events share similar molecular mechanisms, which have been conserved in evolution from protozoa to mammals, and involve common cytoskeletal control mechanisms that can sense the environment, respond to signals, and modulate cell behavior in response1.
An initial stage in cell migration can be the formation of highly dynamic protrusions at the leading edge of the cell. Behind the lamellipodium one can find the lamella, which couples actin to myosin II-mediated contractility and mediates adhesion to the underlying substrate. Lamellipodia are induced by extracellular stimuli such as growth factors, cytokines, and cell adhesion receptors and are driven by actin polymerization, which provides the physical force that pushes the plasma membrane forward2,3. Many signaling and structural proteins have been implicated in this; among them are Rho GTPases, which act coordinately with other signals to activate actin-regulating proteins such as the Arp 2/3 complex, WASP family proteins, and members of the Formin and Spire families in lamellipodia2,4,5.
In addition to actin polymerization, myosin II activity is required for generating contractile forces at the lamellipodium and the anterior lamella. These contractions, also defined as cell-edge retractions, can also result from depolymerization of dendritic actin at the cell periphery and are critical for developing the lamellipodial leading edge and allowing the protrusion to sense the flexibility of the extracellular matrix and other cells and determine the direction of migration6,7,8. Cell edge protrusions that cannot attach to the substrate will form peripheral membrane ruffles, sheet-like structures that appear on the ventral surface of lamellipodia and lamella and move backward relative to the direction of migration. As the lamellipodium fails to attach to the substrate, a posterior lamellipodium forms underneath it and mechanically pushes the first lamellipodium toward the upper ventral surface. The actin filaments in the ruffle that were formerly parallel to the substrate now become perpendicular to it, and the ruffle is now positioned above the advancing lamellipodium. The ruffle that moves backward falls back into the cytosol and represents a cellular mechanism for recycling lamellipodial actin9,10.
Here, we describe an assay for the measurement of cell-edge protrusion dynamics. The protrusion assay uses time-lapse video microscopy to measure single cell-edge protrusion dynamics for 10 min during the spreading phase of the cell. Protrusion dynamics are analyzed by generating kymographs from these movies. In principle, a kymograph imparts detailed quantitative data of moving particles in a spatiotemporal plot to yield a qualitative understanding of cell edge dynamics. The intensity of the moving particle is plotted for all image stacks in a time versus space plot, where the X-axis and the Y-axis represent time and distance, respectively11. This method uses a manual kymograph analysis with ImageJ to get detailed quantitative data, enabling retrieving information from movies and images in case of low signal-to-noise ratio and/or high feature density, and the analysis of images acquired in phase-contrast light microscopy or poor image quality.
The cell-edge protrusion dynamics assay described herein is a fast, simple, and cost-effective method. As such, and because it has been shown to directly correlate with cell migration11,12, it can be used as a preliminary method for testing cytoskeletal dynamics involved in cell motility before deciding to perform more resource-demanding methods. Moreover, it also enables quantitative measurement of how genetic manipulations (knockout, knockdown, or rescue constructs) of cytoskeletal proteins impact cytoskeletal dynamics using a straightforward platform. The assay is an instructive model for exploring cytoskeletal dynamics in the context of cell migration and could be used for elucidation of the mechanisms and molecules underlying cell motility.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm cell culture plates</td>
			<td>Greiner</td>
			<td>P7612-360EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle medium (DMEM)</td>
			<td>Biological Industries, Israel</td>
			<td>01-055-1A</td>
			<td>Medium contains high glucose (4.5 g/L D-glucose)</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate buffered saline (1xDPBS)</td>
			<td>Biological Industries, Israel</td>
			<td>02-023-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Biological Industries, Israel</td>
			<td>04-001-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin from human plasma, liquid, 0.1%, suitable for cell culture</td>
			<td>Sigma-Aldrich</td>
			<td>F0895</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td>Cellvis</td>
			<td>D35-20-1.5-N</td>
			<td>35mm glass bottom dish, dish size 35 mm, well size 20mm, #1.5 cover glass (0.16-0.19 mm).</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>NIH</td>
			<td></td>
			<td>Feely available at: https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr>
			<td>LAS-AF Leica Application Suite 3.2</td>
			<td></td>
			<td></td>
			<td>Microscope acquisition software equipped with an ORCA-Flash 4.0 V2 digital CMOS</td>
		</tr>
		<tr>
			<td>Leica AF6000</td>
			<td>Leica</td>
			<td></td>
			<td>Inverted bright field microscope (40x, NA 1.3 ) equipped with phase-contrast optics, an incubator, and CO2 unit with LAS AF acquisition software equipped with an ORCA-Flash 4.0 V2 digital CMOS camera .</td>
		</tr>
		<tr>
			<td>L-glutamine solution</td>
			<td>Biological Industries, Israel</td>
			<td>03-020-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>ORCA-Flash 4.0 V2 digital CMOS camera</td>
			<td>Hamamatsu Photonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin solution</td>
			<td>Biological Industries, Israel</td>
			<td>03-031-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution B (0.25%), EDTA (0.05%)</td>
			<td>Biological Industries, Israel</td>
			<td>03-052-1A</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring cell-edge protrusion dynamics during spreading using live-cell microscopy

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.

In the experiment described in Figure 2, immortalized MEFs were plated on glass-bottom dishes pre-coated with fibronectin to activate integrin-mediated signaling, blocked by denatured BSA, to block free potential sites for cell adhesion which is not dependent on integrin activation. To reach the logarithmic growth phase at 70%-80% confluence of cells on the day of the experiment, 0.7 x 106 MEFs were plated in a 10 cm diameter tissue culture plate 16 h before the experiment. On the...

Watch this Scientific Journal Video about Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy at JoVE.com

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...

The cell-edge protrusion assay has been shown to directly correlate with cell migration. Therefore it can be used as a preliminary method for identifying critical proteins and signaling mechanisms involved in cell motility. The method is fast, simple, cost effective and does not require fluorescent labeling or using an expensive fluorescent microscope.Demonstrating the procedure will be Michal Gendler, a student from my laboratory. Add two milliliters of one normal hydrochloric acid solution at the center of a glass bottom dish and incubate for 20 minutes at room temperature. Wash the dish three times with two milliliters of PBS.Dilute fibronectin at 10 micrograms per milliliter concentration in PBS and add 200 microliters of the diluted solution to the glass center dish. Incubate for one hour at 37 degrees Celsius. Prepare 1%BSA solution and PBS and pass it through a 0.2 micrometer filter.Denature the solution by incubating at 70 degrees Celsius for 30 minutes in a pre-warmed water bath. Wash the coated glass bottom dish with two milliliters of PBS three times. Add two milliliters of denatured BSA solution and incubate the dish at 37 degree Celsius for one hour.Wash the glass dish with two milliliters of PBS three times. Before 16 to 18 hours of experiment at 0.7 million cells per 10 centimeter diameter tissue culture plate to achieve 70 to 80%confluency. The next day, add two milliliters of trypsin solution to the tissue culture plate and incubate for two to three minutes until the cells get detached.Add five milliliters of the medium to inactivate the trypsin. Using a hemacytometer, count the cells and plate 20, 000 cells in two milliliters of the complete medium in a bottom dish. Then incubate the dish with plated cells in an incubator for 15 minutes.Turn on the heating unit and set it at 37 degrees Celsius one hour before imaging. Also, turn on the carbon dioxide unit and set it at 5%10 minutes before imaging. Switch on the microscope and camera.Turn on the computer and open the microscope acquisition software. Set the magnification on a 40X dry lens phase contrast. Set the total movie duration to 10 minutes with a time interval of five seconds.After 15 minutes of incubation, place the glass bottom dish with adhered cells into an adapter and fix it. Insert the adapter with the dish into the slots in the microscope stage. Take off the dish cover, place the carbon dioxide lid and open the carbon dioxide valve.Locate an appropriate cell for imaging. Start movie acquisition after focusing on the cell. To perform the image analysis, select the straight tool in the image analysis software and make eight lines of 20 arbitrary units perpendicular to the protrusions in a radial arrangement at every 45 degrees, including lamella and cell-edge.In the main toolbar, go to image, select Stacks and then click on Reslice to generate a kymograph picture describing the movement of single points within the cell membrane. Using the kymograph images, extract and manually count the number of protrusions, retractions and ruffles in each of the eight regions in the cell marked by the grid lines. These numbers represent the frequency of protrusions, retractions and ruffles per 10 minutes.Determine the protrusion persistence, distance and velocity by kymography analysis. To determine the protrusion distance, draw perpendicular line from the base of the protrusion to the highest peak of the protrusion. Press M to measure the length of the line in pixels and ensure that the pixel to micrometer ratio is known to convert the length in micrometers.Quantification of protrusions, reactions, and ruffles was performed manually per 10 minutes and the average frequency obtained for protrusions and retractions was 5.1 and 2.1 for ruffles. The representative kymograph had a protrusion distance of approximately 4.8 micrometers and a protrusion time of approximately 0.6 minutes. An example of a cell that should be excluded from the analysis is represented.The kymography analysis revealed that the cell was not present in its spreading phase and did not show any clear membrane protrusions. The most important thing is to choose correct cells for imaging. A proper cell should be in its spreading phase and should not touch other cells to avoid cell-cell communication and signaling.The method can be used as a preliminary tool for testing cytoskeletal dynamics involving cell motility before deciding to perform more results demanding cell migration assays.

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