Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy
November 1st, 2021
This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.
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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Overview
This study presents a cell-edge protrusion assay to investigate the dynamic parameters of spreading cells, including protrusions, retractions, and ruffles. This method is particularly significant as it correlates with cell migration, aiding in the identification of critical proteins and signaling mechanisms involved in cell motility.
Key Study Components
Research Area
- Cell migration
- Cell motility
- Cytoskeletal dynamics
Background
- A correlation exists between cell-edge protrusions and cell migration.
- The assay is straightforward, cost-effective, and does not necessitate fluorescent labeling.
- Understanding protrusions can provide insights into cellular behavior and mechanisms driving motility.
Methods Used
- Cell-edge protrusion assay
- Adhered cells on a glass bottom dish
- Kymography analysis for assessing protrusions
Main Results
- Average frequency of protrusions was 5.1 per 10 minutes, with ruffles at 2.1.
- Kymography analysis revealed a protrusion distance of approximately 4.8 micrometers.
- Identified the importance of choosing appropriate cells in the spreading phase for accurate results.
Conclusions
- The study introduces an accessible assay to assess cell motility dynamics.
- This method serves as a preliminary assessment tool before more complex migration assays are undertaken.
Frequently Asked Questions
Executive Industry Relevance
Cell-edge protrusion dynamics serve as an early, predictive readout of cell motility, enabling rapid assessment of cytoskeletal regulators before committing to resource-intensive migration assays. This method supports target validation by linking molecular perturbations to functional membrane dynamics, providing mechanistic de-risking in discovery workflows. Its label-free, cost-effective design facilitates high-throughput screening of signaling pathways relevant to wound healing, immune response, and cancer metastasis.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Measures protrusion frequency, persistence, distance, and velocity to interrogate therapeutic hypotheses on actin regulators and motility pathways.
- Operational Value: Enables rapid, label-free screening of protein knockdowns or inhibitors affecting cytoskeletal dynamics.
- Predictive Value: Correlates protrusion dynamics with cell migration, supporting go/no-go decisions based on early phenotypic readouts.
Screening & Assay Development
- Scientific Value: Generates quantitative kymograph-derived metrics (protrusion count, distance, velocity) for standardized comparison across experimental conditions.
- Operational Value: Uses phase-contrast microscopy and accessible image analysis tools, reducing dependency on specialized equipment or fluorescent probes.
- Scalability: Supports multi-condition testing via radial sampling of eight membrane regions per cell, enhancing data robustness.
Translational & Preclinical Research
- Translational Continuity: Bridges molecular target modulation to functional cellular behavior in disease-relevant systems like fibroblasts or immune cells.
- Mechanistic De-risking: Clarifies whether observed phenotypic effects stem from altered protrusion dynamics rather than secondary cytotoxicity or adhesion defects.
- Biomarker Alignment: Protrusion parameters can serve as translational biomarkers when linked to in vivo motility or invasion phenotypes.
Pipeline & Workflow Integration
The assay fits within the discovery continuum from target engagement to phenotypic validation, acting as a bridge between biochemical screening and functional cell-based assays.
- Discovery Biology: Tests how genetic or pharmacological manipulations of actin regulators (e.g., Arp2/3, cofilin, Rho GTPases) impact protrusion dynamics as a proxy for motility.
- Screening: Delivers reproducible, quantitative outputs (e.g., protrusions per 10 minutes, protrusion velocity) that enable comparison across compound libraries or genetic screens.
- Analytics: Kymography provides temporal and spatial resolution of membrane dynamics, allowing extraction of kinetic parameters critical for mechanistic modeling.
- Translational Research: Supports continuity to preclinical models by validating that target hits produce consistent protrusion phenotypes in primary or disease-relevant cell lines.
- Enterprise Reuse: Establishes a standardized, low-cost platform for repeated use across projects investigating cell motility in development, inflammation, or oncology.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity by directly linking molecular perturbations to quantifiable membrane dynamics.
- Operational Value: Eliminates need for fluorescent labeling and expensive imaging hardware, lowering assay barrier and increasing accessibility.
- Strategic Value: Improves capital efficiency by filtering targets early using a predictive, phenotypic anchor to cell migration.
- Portfolio Impact: Enables risk-adjusted prioritization of targets based on concordance between protrusion dynamics and migratory potential.
Implementation Considerations
- Requires expertise in live-cell imaging, kymograph generation, and manual or semi-automated protrusion analysis.
- Depends on phase-contrast microscopy and image analysis software capable of reslice and kymograph generation.
- Necessitates standardization of cell seeding density, substrate coating (fibronectin/BSA), and environmental controls (37°C, 5% CO2) for reproducibility.
- Requires training to identify appropriate spreading-phase cells and avoid confluent or contacting cells that confound protrusion measurements.
- Limited to 2D substrate-associated spreading; may not fully capture 3D invasion or confined migration phenotypes.
