Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

Summary

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

Abstract

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Introduction

Animal cells interact physically with the matrix and the other cells that constitute their environment¹. This is required for them to migrate, internalize bodies, acquire external information, or differentiate. In such processes, the cell must generate mechanical forces and, as numerous studies have shown over the recent years, the ability of a cell to generate forces and probe its environment influences its biological behavior, directing for instance proliferation or differentiation^2,3. In turn, the measurement of cellular forces is a major aid to study the regulation of force generati....

Protocol

1. Preparation of Formvar-Coated Grids

1. Clean electron microscopy grids with pure acetone and dry them on filter paper. Then clean a microscope slide with pure ethanol, wipe with a lens paper and remove dust with a blower.
2. Place an ethanol-cleaned glass slide vertically in the funnel of a film casting device containing a solution of Formvar in the lower part. Cover the top of the funnel.
3. Pump 100 mL of Formvar solution (0.5% in ethylene dichloride) with the atomizer bulb until the level reaches two thirds of the slide.
4. Keep the slide in the solution for 1 min.
5. Open the valve of the device to drain the Formvar solution....

Results

The above protocol describes how to prepare the experimental setup to quantify protrusion forces applied by macrophage podosomes on a Formvar substrate. This is achieved using AFM and is illustrated in Figure 1.

When analyzing a topographical image of bulges beneath podosomes using the JPK data processing software, a third-degree polynomial fit should be subtracted from each scan line independently........

Discussion

Material properties

The choice of the material for the deformable membrane, in our case Formvar, needs to fulfill a few requirements. The material must be transparent to visible light and exhibit limited auto fluorescence to allow observations in bright field and fluorescence microscopy. The roughness of the thin film must be well below 10 nm to avoid any topographical effect on cell adhesion and to allow clear observation of the cell-induced protrusions by AFM imaging. Finall.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
200 mesh nickel grids	Electron Microscopy Sciences	G200-Ni
Filter paper	Sigma-Aldrich	1001-055
Microscope slides	Fisher Scientific	10235612
White stickers 26 x 70 mm	Avery	DP033-100
Film casting device with valve in its outlet	Electron Microscopy Sciences	71305-01
Razorblades	Electron Microscopy Sciences	72000
Ethanol	VWR	1.08543.0250
Acetone	VWR	20066.321
Formvar 0.5% solution in ethylene dichloride	Electron Microscopy Sciences	15820
12 mm coverslips	VWR	631-0666
Inverted microscope	Carl Zeiss	Axiovert 200
Atomic Force Microscope	JPK Instruments	NanoWizard III
Temperature-controlled sample holder	JPK Instruments	BioCell
Silicon nitride cantilever with a nominal spring constant of 0.01 N/m	Veeco Instruments	MLCT-AUHW
PBS	Gibco	14190-094
Double-sided adhesive tape	APLI AGIPA	118100
RPMI 1640	Gibco	31870-025
FCS	Sigma-Aldrich	F7524
HEPES	Sigma-Aldrich	H0887
35 mm glass-bottom Petri dishes	WPI	FD35-100