Mario Encinar

Effect of Actin Organization on the Stiffness of Living Breast Cancer Cells Revealed by Peak-Force Modulation Atomic Force Microscopy

We study the correlation between cytoskeleton organization and stiffness of three epithelial breast cancer cells lines with different degrees of malignancy: MCF-10A (healthy), MCF-7 (tumorigenic/noninvasive), and MDA-MB-231 (tumorigenic/invasive). Peak-force modulation atomic force microscopy is used for high-resolution topography and stiffness imaging of actin filaments within living cells. In healthy cells, local stiffness is maximum where filamentous actin is organized as well-aligned stress fibers, resulting in apparent Youngs modulus values up to 1 order of magnitude larger than those in regions where these structures are not observed, but these organized actin fibers are barely observed in tumorigenic cells. We further investigate cytoskeleton conformation in the three cell lines by immunofluorescence confocal microscopy. The combination of both techniques determines that actin stress fibers are present at apical regions of healthy cells, while in tumorigenic cells they appear only at basal regions, where they cannot contribute to stiffness as probed by atomic force microscopy. These results substantiate that actin stress fibers provide a dominant contribution to stiffness in healthy cells, while the elasticity of tumorigenic cells appears not predominantly determined by these structures. We also discuss the effects of the high-frequency indentations inherent to peak-force atomic force microscopy for the identification of mechanical cancer biomarkers. Whereas conventional low loading rate indentations (1 Hz) result in slightly differentiated average stiffness for each cell line, in high-frequency measurements (250 Hz) healthy cells are clearly discernible from both tumorigenic cells with an enhanced stiffness ratio; however, the two cancerous cell lines produced indistinguishable results.

Fabrication and Mechanical Characterization of Semi-Free-Standing (Conjugated) Polymer Thin Films

Polymers undergo severe low-dimensionality effects when they are confined to ultrathin films since most of the structural and dynamical processes involving polymer molecules are correlated to length scales of the order of nanometers. However, the real influence of the size limitation over such processes is often hard to identify as it is masked by interfacial effects. We present the fabrication of a new type of nanostructure consisting of poly[[9-(1-octylnonyl)-9H-carbazole-2,7-diyl]-2,5-thiophenediyl-2,1,3-benzothiadiazole-4,7-diyl-2,5-thiophenediyl] (PCDTBT) thin film that is held up exclusively over tips of poly(ether-ether-ketone) (PEEK) nanopillars. The fabrication method exploits the nonwetting behavior of PCDTBT onto an ordered PEEK nanopillar array when the mobility of the PCDTBT molecules is enhanced by a solvent annealing process. We use this new configuration to characterize the mechanical behavior of free-standing thin film regions, thus in the absence of underlaying substrate, by means of an atomic force microscope (AFM) setup. First, we study how the finite thickness and/or the presence of the underlying substrate influences the mechanical modulus of the material in the linear elastic regime. Moreover, we analyze deep indentations up to the rupture of the thin film, which allow for the measurement of important mechanical features of the nanoconfined polymer, such as its yield strain, the rupture strain, the bending rigidity, etc., which are impossible to investigate in thin films deposited on substrates.

Polymorphism of FtsZ Filaments on Lipid Surfaces: Role of Monomer Orientation

FtsZ is a bacterial cytoskeletal protein involved in cell division. It forms a ringlike structure that attaches to the membrane to complete bacterial division. It binds and hydrolyzes GTP, assembling into polymers in a GTP-dependent manner. To test how the orientation of the monomers affects the curvature of the filaments on a surface, we performed site-directed mutagenesis on the E. coli FtsZ protein to insert cysteine residues at lateral locations to orient FtsZ on planar lipid bilayers. The E93C and S255C mutants were overproduced, purified, and found to be functionally active in solution, as well as being capable of sustaining cell division in vivo in complementation assays. Atomic force microscopy was used to observe the shape of the filament fibers formed on the surface. The FtsZ mutants were covalently linked to the lipids and could be polymerized on the bilayer surface in the presence of GTP. Unexpectedly, both mutants assembled into straight structures. E93C formed a well-defined lattice with monomers interacting at 60° and 120° angles, whereas S255C formed a more open array of straight thicker filament aggregates. These results indicate that filament curvature and bending are not fixed and that they can be modulated by the orientation of the monomers with respect to the membrane surface. As filament curvature has been associated with the force generation mechanism, these results point to a possible role of filament membrane attachment in lateral association and curvature, elements currently identified as relevant for force generation.

Modeling the interplay between protein and lipid aggregation in supported membranes

We present a theoretical model that deals with the complex interplay between lipid segregation and the self-aggregation of lipid-attached proteins. The model, in contrast to previous ones that consider proteins only as passive elements affecting the lipid distribution, describes the system including three terms: the dynamic interactions between protein monomers, the interactions between lipid components, and a mixed term considering protein-lipid interactions. It is used to explain experimental results performed on a well-defined system in which a self-aggregating soluble bacterial cytoskeletal protein polymerizes on a lipid bilayer containing two lipid components. All the elements considered in a previously described protein model, including torsion of the monomers within the filament, are needed to account for the observed filament shapes. The model also points out that lipid segregation can affect the length and curvature of the filaments and that the dynamic behavior of the lipids and proteins can have different time scales, giving rise to memory effects. This simple model that considers a dynamic protein assembly on a fluid and active lipid surface can be easily extended to other biologically relevant situations in which the interplay between protein and lipid aggregation is needed to fully describe the system.

Nanomechanical properties of composite protein networks of erythroid membranes at lipid surfaces

Erythrocyte membranes have been particularly useful as a model for studies of membrane structure and mechanics. Native erythroid membranes can be electroformed as giant unilamellar vesicles (eGUVs). In the presence of ATP, the erythroid membrane proteins of eGUVs rearrange into protein networks at the microscale. Here, we present a detailed nanomechanical study of individual protein microfilaments forming the protein networks of eGUVs when spread on supporting surfaces. Using Peak Force tapping Atomic Force Microscopy (PF-AFM) in liquid environment we have obtained the mechanical maps of the composite lipid-protein networks supported on solid surface. In the absence of ATP, the protein pool was characterized by a Youngs Modulus Epool≈5-15MPa whereas the complex filaments were found softer after protein supramolecular rearrangement; Efil≈0.4MPa. The observed protein softening and reassembling could be relevant for understanding the mechanisms of cytoskeleton reorganization found in pathological erythrocytes or erythrocytes that are affected by biological agents.

FtsZ protein on bilayer membranes: Effects of specific lateral bonds

We use a simple lattice model to explore the self-assembled structures of protein filaments. Monte Carlo simulations show polymorphic structures, formed by the competition between the bundling of filaments, their branching and the spontaneous curvature. In the top-down approach, typical of statistical physics, we compare the simulation results with experimental atomic force microscopy images of the bacterial protein FtsZ anchored on bilayer membranes. We identify the relevant aspects of the interactions between protein monomers, which are amplified as a collective effect of the whole system. From the perspective of soft-matter physics, the experimental system and its model representation provide an interesting example of living polymers with tunable interactions.

Spatially Multiplexed Micro-Spectrophotometry in Bright Field Mode for Thin Film Characterization.

Thickness characterization of thin films is of primary importance in a variety of nanotechnology applications, either in the semiconductor industry, quality control in nanofabrication processes or engineering of nanoelectromechanical systems (NEMS) because small thickness variability can strongly compromise the device performance. Here, we present an alternative optical method in bright field mode called Spatially Multiplexed Micro-Spectrophotometry that allows rapid and non-destructive characterization of thin films over areas of mm2 and with 1 μm of lateral resolution. We demonstrate an accuracy of 0.1% in the thickness characterization through measurements performed on four microcantilevers that expand an area of 1.8 mm2 in one minute of analysis time. The measured thickness variation in the range of few tens of nm translates into a mechanical variability that produces an error of up to 2% in the response of the studied devices when they are used to measure surface stress variations.

Relationship Between Composition, Structure and Dynamics of Main-Chain Liquid Crystalline Polymers with Biphenyl Mesogens

Semiflexible polyesters prepared by an alternating arrangement of biphenyl mesogenic units and aliphatic spacers represent an ideal model for studying the behavior of main-chain liquid crystalline polymers (MCLCP). The transition temperatures, type of mesophase and rate of mesophase–crystal transformation can be tailored by the utilization of a suitable flexible spacer, so that polymers with a stable mesophase at room temperature can be prepared. Furthermore, if the structure of the spacer is adequately chosen, the liquid-crystallization can be slowed down, and sometimes it is possible to quench the amorphous state by cooling the isotropic melt at not very high rates. Following these strategies a rich variety of glass forming liquid crystalline polymers can be prepared. The dynamical behavior of a MCLCP is closely related to the phase present. The segmental dynamics (α relaxation) is the result of cooperative motions near the glass transition of repeating units of the chains built in the structure. The α process behaves differently in the amorphous than in the liquid crystalline (or crystalline) states. Here, the preparation of the polymers and the analysis of their mesomorphic behavior by differential scanning calorimetry and X-ray diffraction are reviewed. The discussion of mechanical/electrical relaxations related to different phases is also included. Eventually, the link between molecular composition, structure and dynamics of the MCLCPs will be established.