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

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

CNRS, LAAS

Univ de Toulouse, INSA

A critical step in the fabrication of biochips is the controlled placement of probes molecules on solid surfaces. This is currently performed by sequential deposition of probes on a target surface with split or solid pins. In this article, we present a cost-effective procedure namely microcontact printing using stamps, for a parallel deposition of probes applicable for manufacturing biochips.

Direct microcontact printing of oligonucleotides for biochip applications.

It is well-established that, during microcontact printing (muCP) using poly(dimethylsiloxane) (PDMS)-based stamps, some unexpected siloxane fragments can be transferred from the stamp to the surface of the sample. This so-called contamination effect coexists with the delivery of the molecules constituting the ink and by this way influences the printing process. The real impact of this contamination for the muCP technique is still partially unknown. In this work, we investigate the kinetics of this contamination process through the surface characterization of both the sample and the stamp after imprinting. The way both the curing conditions of the PDMS material and the contact time influence the degree of contamination of the surface is investigated on silicon and glass substrates. We propose a cleaning process of the stamp during several hours which eliminates any trace of contamination during printing. We show that hydrophobicity recovery of PDMS surfaces after hydrophilic treatment using oxygen plasma is considerably slowed down when the PDMS material is cleaned using our procedure. Finally, by comparing cleaned and uncleaned PDMS stamps, we show the influence of contamination on the quality of muCP using fluorescent DNA molecules as an ink. Surprisingly, we observe that the amount of DNA molecules transferred during muCP is higher for the uncleaned stamp, highlighting the positive impact of the presence of low molecular weight siloxane fragments on the muCP process. This result is attributed to the better adsorption of oligonucleotides on the stamp surface in presence of these contaminating molecules.

Poly(dimethylsiloxane) contamination in microcontact printing and its influence on patterning oligonucleotides.

Microtransfer molding has been used to fabricate homogeneous micropatterns and nanopatterns of spin crossover nanoparticles of [Fe(NH(2)trz)](tos)(2) over a large area. We show that the use of an aprotic solvent (n-octane) may lead to successful results. Very well organized micropatterns are obtained, showing spin crossover phenomenon. Dark field optical and AFM images and Raman microspectrometry results are reported.

Soft lithographic patterning of spin crossover nanoparticles.

Podosomes are adhesion structures formed in monocyte-derived cells. They are F-actin-rich columns perpendicular to the substrate surrounded by a ring of integrins. Here, to measure podosome protrusive forces, we designed an innovative experimental setup named protrusion force microscopy (PFM), which consists in measuring by atomic force microscopy the deformation induced by living cells onto a compliant Formvar sheet. By quantifying the heights of protrusions made by podosomes onto Formvar sheets, we estimate that a single podosome generates a protrusion force that increases with the stiffness of the substratum, which is a hallmark of mechanosensing activity. We show that the protrusive force generated at podosomes oscillates with a constant period and requires combined actomyosin contraction and actin polymerization. Finally, we elaborate a model to explain the mechanical and oscillatory activities of podosomes. Thus, PFM shows that podosomes are mechanosensing cell structures exerting a protrusive force. 

Protrusion force microscopy reveals oscillatory force generation and mechanosensing activity of human macrophage podosomes.

Podosomes are mechanosensitive adhesion cell structures that are capable of applying protrusive forces onto the extracellular environment. We have recently developed a method dedicated to the evaluation of the nanoscale forces that podosomes generate to protrude into the extracellular matrix. It consists in measuring by atomic force microscopy (AFM) the nanometer deformations produced by macrophages on a compliant Formvar membrane and has been called protrusion force microscopy (PFM). Here we perform time-lapse PFM experiments and investigate spatial correlations of force dynamics between podosome pairs. We use an automated procedure based on finite element simulations that extends the analysis of PFM experimental data to take into account podosome architecture and organization. We show that protrusion force varies in a synchronous manner for podosome first neighbors, a result that correlates with phase synchrony of core F-actin temporal oscillations. This dynamic spatial coordination between podosomes suggests a short-range interaction that regulates their mechanical activity. 

Working together: spatial synchrony in the force and actin dynamics of podosome first neighbors.

Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment. However, how cellular forces are distributed to allow this protruding mechanism is still unknown. To investigate the molecular machinery of protrusion force generation, we performed mechanical simulations and developed quantitative image analyses of nanoscale architectural and mechanical measurements. First, in silico modeling showed that the deformations of the substrate made by podosomes require protrusion forces to be balanced by local traction forces at the immediate core periphery where the adhesion ring is located. Second, we showed that three-ring proteins are required for actin polymerization and protrusion force generation. Third, using DONALD, a 3D nanoscopy technique that provides 20 nm isotropic localization precision, we related force generation to the molecular extension of talin within the podosome ring, which requires vinculin and paxillin, indicating that the ring sustains mechanical tension. Our work demonstrates that the ring is a site of tension, balancing protrusion at the core. This local coupling of opposing forces forms the basis of protrusion and reveals the podosome as a nanoscale autonomous force generator.

Podosome Force Generation Machinery: A Local Balance between Protrusion at the Core and Traction at the Ring.

We present a new strategy for fabricating a silicon nanopore device allowing straightforward fluidic integration and electrical as well as optical monitoring. The device presents nanopores of diameters 10 nm to 160 nm, and could therefore be used to obtain solvent-free free-standing lipid bilayers from small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV). The silicon chip fabrication process only requires front side processing of a silicon-on-insulator (SOI) substrate. A polydimethylsiloxane (PDMS) microfluidic interface is assembled on the silicon chip for fluidic handling and electrical addressing. We detail the electrical specifications of our device and some perspectives showing that the use of an SOI substrate is a convenient way to reduce the electrical noise in a silicon nanopore device without the need of a photolitographic patterned passivation layer. We then demonstrate simultaneous electrical and optical monitoring by capturing negatively charged fluorescent nanoparticles. Finally, in the perspective of solvent-free free-standing lipid bilayers, we show that incubation of SUV results in a drastic increase of the device electrical resistance, which is likely due to the formation of a free-standing lipid bilayer sealing the nanopores. Graphical abstract ᅟ.

Christophe Thibault

CNRS, LAAS

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