Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers

The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression. Here, we explored relevant biophysical properties of vimentin IFs in living cells combining confocal microscopy and a filament tracking routine that allows localizing filaments with ~20 nm precision. A Fourier-based analysis showed that IFs curvatures followed a thermal-like behavior characterized by an apparent persistence length (lp*) similar to that measured in aqueous solution. Additionally, we determined that certain perturbations of the cytoskeleton affect lp* and the lateral mobility of IFs as assessed in cells in which either the microtubule dynamic instability was reduced or actin filaments were partially depolymerized. Our results provide relevant clues on how vimentin IFs mechanically couple with microtubules and actin filaments in cells and support a role of this network in the response to mechanical stress.Fil: Smoler, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Coceano, Giovanna. Royal Institute of Technology; SueciaFil: Testa, Ilaria. Royal Institute of Technology; SueciaFil: Bruno, Luciana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; ArgentinaFil: Levi, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin

Glucose is a key driver for GLUT1-mediated nanoparticles internalization in breast cancer cells

The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy

Vimentin Levels and Serine 71 Phosphorylation in the Control of Cell-Matrix Adhesions, Migration Speed, and Shape of Transformed Human Fibroblasts

Metastasizing tumor cells show increased expression of the intermediate filament (IF) protein vimentin, which has been used to diagnose invasive tumors for decades. Recent observations indicate that vimentin is not only a passive marker for carcinoma, but may also induce tumor cell invasion. To clarify how vimentin IFs control cell adhesions and migration, we analyzed the nanoscale (30–50 nm) spatial organization of vimentin IFs and cell-matrix adhesions in metastatic fibroblast cells, using three-color stimulated emission depletion (STED) microscopy. We also studied whether wild-type and phospho-deficient or -mimicking mutants of vimentin changed the size and lifetime of focal adhesions (FAs), cell shape, and cell migration, using live-cell total internal reflection imaging and confocal microscopy. We observed that vimentin exists in fragments of different lengths. Short fragments were mostly the size of a unit-length filament and were mainly localized close to small cell-matrix adhesions. Long vimentin filaments were found in the proximity of large FAs. Vimentin expression in these cells caused a reduction in FAs size and an elongated cell shape, but did not affect FA lifetime, or the speed or directionality of cell migration. Expression of a phospho-mimicking mutant (S71D) of vimentin increased the speed of cell migration. Taken together, our results suggest that in highly migratory, transformed mesenchymal cells, vimentin levels control the cell shape and FA size, but not cell migration, which instead is linked to the phosphorylation status of S71 vimentin. These observations are consistent with the possibility that not only levels, but also the assembly status of vimentin control cell migration

LOCAL MEASUREMENT OF BREAST CANCER CELLS MECHANICAL PROPERTIES

2013/2014In the last decades cell mechanics has been increasingly associated to cell health and function. Elasticity is one of the most investigated mechanical properties of cells and is now considered as a potential label free marker of cancer progression. In this Thesis I report on the characterization of cells based on their mechanical properties. Three different biophysical micromanipulation tools have been used: Optical Tweezers (OT), Atomic Force Microscopy (AFM) and Speckle Sensing Microscopy (SSM). We chose three breast cell lines selected as a model to study cancer progression: MDA-MB-231, a highly aggressive cell line belonging to the Basal cell-like phenotype; MCF-7, a less aggressive tumour cell line, belonging to the Luminal A cell-like tumour subtype; and HBL-100, a non neoplastic cell line, derived from the milk of a Caucasian woman, normal control for breast basal-myoepithelial cells. The viscoelastic properties of the three cell lines have been measured using complementary approaches, thus allowing a thorough characterization: OT membrane tether pulling, OT and AFM vertical cell indentation and speckle interferometry with SSM. With AFM and OT techniques we performed local measurements on specific parts of the cell; while with SSM we considered the cell as a whole viscoelastic body and we analyzed groups of cells at the same time. OT membrane tether pulling uses a microbead trapped by the laser beam to pull cellular membrane tethers; from the resultant Force-Elongation (FE) curve, some viscoelastic parameters of the cell itself have been extracted and compared. The experimental approach results to be inefficient and time consuming and it has been, therefore, substituted by OT vertical indentation. The new approach uses the OT in a similar way of the AFM technique, i.e. indenting the cell with a micron sized bead trapped by the laser. The elastic modulus has been therefore measured by vertical cell indentation, employing AFM and OT as two complementary techniques: with AFM we applied nN forces at high loading rates, while with OT we operated at pN forces at low loading rates. OT has been implemented in an inverted optical microscope and the elastic modulus of the three cell lines results to be: 23.4 (HBL-100), 31.2 (MCF-7) and 12.6 (MDA-MB-231) Pa. AFM indentation approach has been performed using the Bioscope Catalyst in Peak Force Quantitative Nanomechanical Mapping (PF-QNM) mode. Bioscope is able of applying nN forces by means of a nano-sized tip attached at the end of a cantilever. This new AFM mode allows mapping different mechanical properties of the cell under scan. The elastic modulus of the three cell lines has been extracted, providing more information about the mechanical alterations undergoing tumorigenesis. The mean values measured near the cell nucleus were: 91.1 (HBL-100), 81.8 (MCF-7), 57.6 (MDA-MB-231) kPa. These results show that there is an inverse correlation between cell stiffness and breast cancer cell aggressiveness, since MDA-MB-231, the most aggressive cell line, has an elastic modulus significantly lower than the other two cell lines, both with OT and AFM measurements. The difference values obtained by AFM and OT are the result of the different regimes used by these techniques: AFM applies higher forces and higher loading rates in comparison to OT. Nevertheless, the trend of the values between the cell lines was the same, showing that the aggressive cells were much softer than the other two. The combination of the two techniques is proposed for a more complete characterization of the mechanical properties of cells in different mechanical conditions. Moreover we show that the stiffness of the substrate influences the elasticity of the cells; OT vertical indentation has been applied to HBL-100 cells cultured on bare and collagen coated substrates and their elastic modulus was 26±9 for bare and 19±7 Pa for collagen. These results show that cells adapt their structures to that of the substrate and demonstrate the potential of this setup for low-force probing of cell mechanics. SSM has been originally proposed by our group in an international collaboration for fast diagnosis of malaria making available the analysis of thousand of cells per minute. It is based on the analysis of the speckles formed by light scattered by the cells when illuminated by a tilted laser beam. Speckle dynamics reflects the thermal vibration of the cell, which is linked to its stiffness. In this work SSM has been applied to MCF 7 cell line for cell mechanics characterization. The final goal of this PhD Thesis is the characterization of the mechanical properties of cancer cells, by means of an integrated method based on rigorous biophysical techniques to understand the disease progression and differentiation towards metastasis.XXVII Ciclo198

Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems.

The theoretically unlimited spatial resolution of fluorescence nanoscopy often comes at the expense of time, contrast and increased dose of energy for recording. Here, we developed MoNaLISA, for Molecular Nanoscale Live Imaging with Sectioning Ability, a nanoscope capable of imaging structures at a scale of 45-65 nm within the entire cell volume at low light intensities (W-kW cm-2). Our approach, based on reversibly switchable fluorescent proteins, features three distinctly modulated illumination patterns crafted and combined to gain fluorescence ON-OFF switching cycles and image contrast. By maximizing the detected photon flux, MoNaLISA enables prolonged (40-50 frames) and large (50 × 50 µm2) recordings at 0.3-1.3 Hz with enhanced optical sectioning ability. We demonstrate the general use of our approach by 4D imaging of organelles and fine structures in epithelial human cells, colonies of mouse embryonic stem cells, brain cells, and organotypic tissues

Highly IR-transparent microfluidic chip with surface-modified BaF2 optical windows for Infrared Microspectroscopy of living cells

In this contribution we present the first example of a microfluidic chip based on BaF2for Infrared Microspectroscopy (IRMS) of living cells. The advantage in using barium fluoride as platform relies on its high IR transparency, especially in the spectral region below 1300 cm 1 , where the absorption bands of nucleic acids and carbohydrates are located. Barium fluoride is slightly soluble in water (0.12 g/100 g water) and it is potentially harmful for living cells. To overcome these problems, here we exploit an approach whose feasibility has been demonstrated previously on CaF2: the surface modification obtained by sputtering a thin Si layer on the surface. The Surface Modified Microfluidic Devices (SM-MD) hence obtained not only solve the BaF2drawbacks, but also provide a silicon-like substrate fully compatible with standard micro-fabrication processes. These potentialities are here further explored in the direction of chemical or topographical nano-patterning of the silicon-like surface. The silicon thin layer was structured in the shape of 300 nm wide grooves (500 nm pitch) with a thickness of 35 nm by using standard NIL and etching processes; chemical patterning was achieved by exploiting silane chemistry. Finally, we tested the performances of these devices at SISSI beamline@Elettra, collecting IR spectra of single MDA-MB-231 living cells maintained either in physiological solution or complete medium. A comparison of the spectra of a single cell obtained in BaF2and CaF2MDs is reporte

Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems

Super-resolution microscopy often suffers from low contrast and slow recording times. Here the authors present an optical implementation which makes the fluorescent proteins’ ON–OFF switching cycles more efficient, enhancing contrast and spatio-temporal resolution in 3D cell and tissue imaging

RecA finds homologous DNA by reduced dimensionality search

Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3' single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms

Substrate-dependent cell elasticity measured by optical tweezers indentation

In the last decade, cell elasticity has been widely investigated as a potential label free indicator for cellular alteration in different diseases, cancer included. Cell elasticity can be locally measured by pulling membrane tethers, stretching or indenting the cell using optical tweezers. In this paper, we propose a simple approach to perform cell indentation at pN forces by axially moving the cell against a trapped microbead. The elastic modulus is calculated using the Hertz-model. Besides the axial component, the setup also allows us to examine the lateral cell-bead interaction. This technique has been applied to measure the local elasticity of HBL-100 cells, an immortalized human cell line, originally derived from the milk of a woman with no evidence of breast cancer lesions. In addition, we have studied the influence of substrate stiffness on cell elasticity by performing experiments on cells cultured on two substrates, bare and collagen-coated, having different stiffness. The mean value of the cell elastic modulus measured during indentation was 26\ub19 Pa for the bare substrate, while for the collagen-coated substrate it diminished to 19\ub17 Pa. The same trend was obtained for the elastic modulus measured during the retraction of the cell: 23\ub110 Pa and 13\ub17 Pa, respectively. These results show the cells adapt their stiffness to that of the substrate and demonstrate the potential of this setup for low-force probing of modifications to cell mechanics induced by the surrounding environment (e.g. extracellular matrix or other cells)