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* These authors contributed equally
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 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.
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 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's ability to move through 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.
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 segmentation, we recommend using fluorescent markers that are evenly distributed in the cytoplasm, e.g., cytosolic GFP.
2. Drug Incubation and Cell Recovery
3. Magnetic Chamber Preparation
4. Image Acquisition
5. Analysis of cell area, circularity and protrusion dynamics during cell spreading
6. Quantify cell edge dynamics during cell spreading using kymographs
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...
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...
The authors have nothing to disclose.
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.
Name | Company | Catalog Number | Comments |
0.05% Trypsin (0.05%), 0.53 mM EDTA | Wisent Bioproducts | 325-042-CL | |
10.0 cm Petri Dish, Polystyrene, TC Treated, Vented | Starstedt | 83.3902 | |
15 mL High Clarity PP Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Sterile | Falcon | 352097 | |
1-Well Chamlide CMS for 22 mm x 22 mm Coverslip | Quorum Technologies | CM-S22-1 | |
35 mm TC-treated Easy-Grip Style Cell Culture Dish | Falcon | 353001 | |
50 mL Centrifuge Tube, Transparent, Plug Seal | Nest | 602002 | |
6.0 cm Cell Culture Dishes Treated for Increased Cell Attachment, Sterile | VWR | 10861-658 | |
Arp2/3 Complex Inhibitor I, CK-666 | Millipore Sigma | 182515 | |
Camera, Prime 95B-25MM | Photometrics | ||
Dimethyl Sulfoxide, Sterile | BioShop | DMS666 | |
DMEM, 1x, 4.5 g/L Glucose, with L-Glutamine, Sodium Pyruvate and Phenol Red | Wisent Bioproducts | 319-005 CL | |
DMEM/F-12, HEPES, No Phenol Red | Gibco | 11039021 | |
D-PBS, 1X | Wisent Bioproducts | 311-425 CL | |
Fetal Bovine Serum | Wisent Bioproducts | 080-110 | |
Fiji Software | ImageJ | ||
HEPES (1 M) | Gibco | 15630080 | |
Human Plasma Fibronectin Purified Protein 1 mg | Millipore Sigma | FC010 | |
Immersion Oil | Cargille | 16241 | |
L-Glutamine Solution (200 mM) | Wisent Bioproducts | 609-065-EL | |
MEM Non-Essential Amino Acids Solution (100X) | Gibco | 11140050 | |
Micro Cover Glasses, Square, No. 11/2 22 x 22 mm | VWR | CA48366-227-1 | |
Microscope Body, Eclipse Ti2-E | Nikon | ||
Objective, CFI Plan Apo Lambda 60X Oil | Nikon | MRD01605 | |
Penicillin-Streptomycin | Sigma | P4333 | |
Spinning Disk, Crest Light V2 | CrestOptics | ||
Spyder | Anaconda | ||
Stage top incubator | Tokai Hit | ||
Statistics Software, Prism | GraphPad | ||
Tweezers, Style 2 | Electron Microscopy Sciences | 78326-42 |
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