Alfredo Celedon

A distinctive role for focal adhesion proteins in three- dimensional cell motility

Focal adhesions are large multi-protein assemblies that form at the basal surface of cells on planar dishes, and that mediate cell signalling, force transduction and adhesion to the substratum. Although much is known about focal adhesion components in two-dimensional (2D) systems, their role in migrating cells in a more physiological three-dimensional (3D) matrix is largely unknown. Live-cell microscopy shows that for cells fully embedded in a 3D matrix, focal adhesion proteins, including vinculin, paxillin, talin, α-actinin, zyxin, VASP, FAK and p130Cas, do not form aggregates but are diffusely distributed throughout the cytoplasm. Despite the absence of detectable focal adhesions, focal adhesion proteins still modulate cell motility, but in a manner distinct from cells on planar substrates. Rather, focal adhesion proteins in matrix-embedded cells regulate cell speed and persistence by affecting protrusion activity and matrix deformation, two processes that have no direct role in controlling 2D cell speed. This study shows that membrane protrusions constitute a critical motility/matrix-traction module that drives cell motility in a 3D matrix.

Mapping Local Matrix Remodeling Induced by a Migrating Tumor Cell Using Three-Dimensional Multiple-Particle Tracking

Mesenchymal cell migration through a three-dimensional (3D) matrix typically involves major matrix remodeling. The direction of matrix deformation occurs locally in all three dimensions, which cannot be measured by current techniques. To probe the local, 3D, real-time deformation of a collagen matrix during tumor cell migration, we developed an assay whereby matrix-embedded beads are tracked simultaneously in all three directions with high resolution. To establish a proof of principle, we investigated patterns of collagen I matrix deformation near fibrosarcoma cells in the absence and presence of inhibitors of matrix metalloproteinases and acto-myosin contractility. Our results indicate that migrating cells show patterns of local matrix deformation toward the cell that are symmetric in magnitude with respect to the axis of cell movement. In contrast, patterns of matrix release from the cell are asymmetric: the matrix is typically relaxed first at the back of the cell, allowing forward motion, and then at the cells leading edge. Matrix deformation in regions of the matrix near the cells leading edge is elastic and mostly reversible, but induces irreversible matrix rupture events near the trailing edge. Our results also indicate that matrix remodeling spatially correlates with protrusive activity. This correlation is mediated by myosin II and Rac1, and eliminated after inhibition of pericellular proteolysis or ROCK. We have developed an assay based on high-resolution 3D multiple-particle tracking that allows us to probe local matrix remodeling during mesenchymal cell migration through a 3D matrix and simultaneously monitor protrusion dynamics.

Magnetic tweezers measurement of single molecule torque.

Torsional stress in linear biopolymers such as DNA and chromatin has important consequences for nanoscale biological processes. We have developed a new method to directly measure torque on single molecules. Using a cylindrical magnet, we manipulate a novel probe consisting of a nanorod with a 0.1 microm ferromagnetic segment coupled to a magnetic bead. We achieve controlled introduction of turns into the molecule and precise measurement of torque and molecule extension as a function of the number of turns at low pulling force. We show torque measurement of single DNA molecules and demonstrate for the first time measurements of single chromatin fibers.

The distinct roles of the nucleus and nucleus-cytoskeleton connections in three-dimensional cell migration

Cells often migrate in vivo in an extracellular matrix that is intrinsically three-dimensional (3D) and the role of actin filament architecture in 3D cell migration is less well understood. Here we show that, while recently identified linkers of nucleoskeleton to cytoskeleton (LINC) complexes play a minimal role in conventional 2D migration, they play a critical role in regulating the organization of a subset of actin filament bundles – the perinuclear actin cap - connected to the nucleus through Nesprin2giant and Nesprin3 in cells in 3D collagen I matrix. Actin cap fibers prolong the nucleus and mediate the formation of pseudopodial protrusions, which drive matrix traction and 3D cell migration. Disruption of LINC complexes disorganizes the actin cap, which impairs 3D cell migration. A simple mechanical model explains why LINC complexes and the perinuclear actin cap are essential in 3D migration by providing mechanical support to the formation of pseudopodial protrusions.

Magnetic Manipulation of Nanorods in the Nucleus of Living Cells

The organization of chromatin in the cell nucleus is crucial for gene expression regulation. However, physically probing the nuclear interior is challenging because high forces have to be applied using minimally invasive techniques. Here, magnetic nanorods embedded in the nucleus of living cells are subjected to controlled rotational forces, producing micron-sized displacements in the nuclear interior. The resulting time-dependent rotation of the nanorods is analyzed in terms of viscoelastic parameters of the nucleus, in wild-type and Lamin A/C deficient cells. This method and analysis reveal that Lamin A/C knockout, together perhaps with other changes that result from the knockout, induce significant decreases in the nuclear viscosity and elasticity.

Torsional Mechanics of DNA Are Regulated by Small-Molecule Intercalation

Whether the bend and twist mechanics of DNA molecules are coupled is unclear. Here, we report the direct measurement of the resistive torque of single DNA molecules to study the effect of ethidium bromide (EtBr) intercalation and pulling force on DNA twist mechanics. DNA molecules were overwound and unwound using recently developed magnetic tweezers where the molecular resistive torque was obtained from Brownian angular fluctuations. The effect of EtBr intercalation on the twist stiffness was found to be significantly different from the effect on the bend persistence length. The twist stiffness of DNA was dramatically reduced at low intercalator concentration (<10 nM); however, it did not decrease further when the intercalator concentration was increased by 3 orders of magnitude. We also determined the dependence of EtBr intercalation on the torque applied to DNA. We propose a model for the elasticity of DNA base pairs with intercalated EtBr molecules to explain the abrupt decrease of twist stiffness at low EtBr concentration. These results indicate that the bend and twist stiffnesses of DNA are independent and can be differently affected by small-molecule binding.

Local mechanical response of cells to the controlled rotation of magnetic nanorods

The mechanical response of the cytoplasm was investigated by the intracellular implantation of magnetic nanorods and exposure to low-frequency rotatory magnetic fields. Nanorods (Pt-Ni, ∼200 nm diameter) fabricated by electrodeposition in templates of porous alumina with lengths of approximately 2 and 5 µm were inserted into NIH/3T3 fibroblasts and manipulated with a rotational magnetic field. Nanorod rotation was observed only for torques greater than 3.0 × 10(-16) Nm, suggesting a Bingham-type behavior of the cytoplasm. Higher torques produced considerable deformation of the intracellular material. The cell nucleus and cell membrane were significantly deformed by nanorods actuated by 4.5 × 10(-15) Nm torques. Our results demonstrate that nanorods under magnetic fields are an effective tool to mechanically probe the intracellular environment. We envision that our findings may contribute to the noninvasive and direct mechanical characterization of the cytoplasm.

Intracellular accumulation and dissolution of silver nanoparticles in L-929 fibroblast cells using live cell time-lapse microscopy

Abstract Cytotoxicity assessments of nanomaterials, such as silver nanoparticles, are challenging due to interferences with test reagents and indicators as well uncertainties in dosing as a result of the complex nature of nanoparticle intracellular accumulation. Furthermore, current theories suggest that silver nanoparticle cytotoxicity is a result of silver nanoparticle dissolution and subsequent ion release. This study introduces a novel technique, nanoparticle associated cytotoxicity microscopy analysis (NACMA), which combines fluorescence microscopy detection using ethidium homodimer-1, a cell permeability marker that binds to DNA after a cell membrane is compromised (a classical dead-cell indicator dye), with live cell time-lapse microscopy and image analysis to simultaneously investigate silver nanoparticle accumulation and cytotoxicity in L-929 fibroblast cells. Results of this method are consistent with traditional methods of assessing cytotoxicity and nanoparticle accumulation. Studies conducted on 10, 50, 100 and 200 nm silver nanoparticles reveal size dependent cytotoxicity with particularly high cytotoxicity from 10 nm particles. In addition, NACMA results, when combined with transmission electron microscopy imaging, reveal direct evidence of intracellular silver ion dissolution and possible nanoparticle reformation within cells for all silver nanoparticle sizes.

Intra- and Extracellular Microrheology of Endothelial Cells in a 3D Matrix

Limited progress in our understanding of molecular cell functions in 3D is due, in part, to the lack of quantitative assays to probe cells in the physiological three-dimensional (3D) milieu. We have recently developed quantitative methods to probe the micromechanical properties of live cells inside a 3D matrix and monitor the dynamics of localized matrix remodeling during 3D cell motility. Conventional biophysical methods are unsuitable for cells fully embedded inside a matrix because these methods require direct physical contact between the probe (i.e. AFM cantilever, magnetic bead, or micropipette tip) and the cell surface. To measure local intracellular micromechanics in live cells, the spontaneous movements of submicron beads lodged in the cytoplasm of the matrix-embedded cells are monitored at a distance with nanometer resolution. The mean squared displacements of the beads are transformed into elastic and viscous moduli, which describe the propensity of the cytoplasm to flow or resist shear stresses. To map extracellular matrix remodeling, the cell-mediated movements of large beads tightly embedded in the matrix are monitored around individual cells in 3D. Here we describe the fundamentals and use of both particle-tracking intracellular microrheology and particle-tracking matrix traction mechanics to probe changes in the micromechanical properties of individual human endothelial cells embedded in 3D matrix and subjected to biochemical stimuli or pharmacological treatments compared with the response of cells on conventional flat substrates.

Magnetic Tweezers Measurement of Single Molecule Torque

Helical duplex DNA continually experiences torque from translocating macromolecular complexes and helix unwinding proteins. We have developed a magnetic tweezers methodology using a cylindrical magnet and magnetic nanorods to directly measure torsional stress, or resistive torque, as twists are introduced at low pulling forces. We demonstrate the utility of this method by measuring the resistive torque of single DNA molecules and, for the first time, single chromatin fibers.Figure: New and conventional magnetic tweezers configurations. (a) In conventional magnetic tweezers, the field orients the induced dipole of the superparamagnetic bead horizontally, producing a strong horizontal angular trap that prevents angular fluctuations of the probe. (b) In the new configuration consisting of a vertical magnetic field and nanorod-bead construct, the magnetic field and the probe dipole align vertically, thus horizontal angular movements are not constrained. A weak horizontal force generates a weak horizontal angular trap allowing us to measure the torque applied to the molecule. (c) Scanning electron micrograph of magnetic Ni-Pt nanorods (bar = 1μm). (d) Bright-field image of nanorod-bead probe. Nanorod and bead self-assemble by magnetic attraction.