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

Summary

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

Abstract

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.

Introduction

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.<....

Protocol

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 C57BL/6 mice. Primary MEFs were generated according to the Jacks laboratory protocol¹³. Cells from five different embryos were p....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 cm cell culture plates	Greiner	P7612-360EA
Bovine serum albumin (BSA)	Sigma-Aldrich	A7906
Dulbecco’s modified Eagle medium (DMEM)	Biological Industries, Israel	01-055-1A	Medium contains high glucose (4.5 g/L D-glucose)
Dulbecco’s phosphate buffered saline (1xDPBS)	Biological Industries, Israel	02-023-1A
Fetal bovine serum (FBS)	Biological Industries, Israel	04-001-1A
Fibronectin from human plasma, liquid, 0.1%, suitable for cell culture	Sigma-Aldrich	F0895
Glass bottom dishes	Cellvis	D35-20-1.5-N	35mm glass bottom dish, dish size 35 mm, well size 20mm, #1.5 cover glass (0.16-0.19 mm).
ImageJ software	NIH		Feely available at: https://imagej.nih.gov/ij/download.html
LAS-AF Leica Application Suite 3.2			Microscope acquisition software equipped with an ORCA-Flash 4.0 V2 digital CMOS
Leica AF6000	Leica		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 .
L-glutamine solution	Biological Industries, Israel	03-020-1B
ORCA-Flash 4.0 V2 digital CMOS camera	Hamamatsu Photonics
Penicillin-streptomycin solution	Biological Industries, Israel	03-031-1B
Trypsin-EDTA solution B (0.25%), EDTA (0.05%)	Biological Industries, Israel	03-052-1A