Published: May 22nd, 2021
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 protrusio....
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.......
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.......
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.....
|0.05% Trypsin (0.05%), 0.53 mM EDTA
|10.0 cm Petri Dish, Polystyrene, TC Treated, Vented
|15 mL High Clarity PP Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Sterile
|1-Well Chamlide CMS for 22 mm x 22 mm Coverslip
|35 mm TC-treated Easy-Grip Style Cell Culture Dish
|50 mL Centrifuge Tube, Transparent, Plug Seal
|6.0 cm Cell Culture Dishes Treated for Increased Cell Attachment, Sterile
|Arp2/3 Complex Inhibitor I, CK-666
|Camera, Prime 95B-25MM
|Dimethyl Sulfoxide, Sterile
|DMEM, 1x, 4.5 g/L Glucose, with L-Glutamine, Sodium Pyruvate and Phenol Red
|DMEM/F-12, HEPES, No Phenol Red
|Fetal Bovine Serum
|HEPES (1 M)
|Human Plasma Fibronectin Purified Protein 1 mg
|L-Glutamine Solution (200 mM)
|MEM Non-Essential Amino Acids Solution (100X)
|Micro Cover Glasses, Square, No. 11/2 22 x 22 mm
|Microscope Body, Eclipse Ti2-E
|Objective, CFI Plan Apo Lambda 60X Oil
|Spinning Disk, Crest Light V2
|Stage top incubator
|Statistics Software, Prism
|Tweezers, Style 2
|Electron Microscopy Sciences
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