Baldomero Alonso-Latorre

Spatio-temporal analysis of eukaryotic cell motility by improved force cytometry

Cell motility plays an essential role in many biological systems, but precise quantitative knowledge of the biophysical processes involved in cell migration is limited. Better measurements are needed to ultimately build models with predictive capabilities. We present an improved force cytometry method and apply it to the analysis of the dynamics of the chemotactic migration of the amoeboid form of Dictyostelium discoideum. Our explicit calculation of the force field takes into account the finite thickness of the elastic substrate and improves the accuracy and resolution compared with previous methods. This approach enables us to quantitatively study the differences in the mechanics of the migration of wild-type (WT) and mutant cell lines. The time evolution of the strain energy exerted by the migrating cells on their substrate is quasi-periodic and can be used as a simple indicator of the stages of the cell motility cycle. We have found that the mean velocity of migration v and the period of the strain energy T cycle are related through a hyperbolic law v = L/T, where L is a constant step length that remains unchanged in mutants with adhesion or contraction defects. Furthermore, when cells adhere to the substrate, they exert opposing pole forces that are orders of magnitude higher than required to overcome the resistance from their environment.

Mesenchymal stem cell durotaxis depends on substrate stiffness gradient strength

Mesenchymal stem cells (MSCs) respond to the elasticity of their environment, which varies between and within tissues. Stiffness gradients within tissues can result from pathological conditions, but also occur through normal variation, such as in muscle. MSC migration can be directed by shallow stiffness gradients before differentiating. Gradients with fine control over substrate compliance - both in range and rate of change (strength) - are needed to better understand mechanical regulation of MSC migration in normal and diseased states. We describe polyacrylamide stiffness gradient fabrication using three distinct systems, generating stiffness gradients of physiological (1 Pa/μm), pathological (10 Pa/μm), and step change (≥ 100Pa/μm) strength. All gradients spanned a range of physiologically relevant elastic moduli for soft tissues (1-12 kPa). MSCs migrated to the stiffest region on each gradient. Time-lapse microscopy revealed that migration velocity correlated directly with gradient strength. Directed migration was reduced in the presence of the contractile agonist lysophosphatidic acid (LPA) and cytoskeleton-perturbing drugs nocodazole and cytochalasin. LPA- and nocodazole-treated cells remained spread and protrusive on the substrate, while cytochalasin-treated cells did not. Nocodazole-treated cells spread in a similar manner to untreated cells, but exhibited greatly diminished traction forces. These data suggest that a functional actin cytoskeleton is required for migration whereas microtubules are required for directed migration. The data also imply that, in vivo, MSCs may preferentially accumulate in regions of high elastic modulus and make a greater contribution to tissue repairs in these locations.

Myosin II Is Essential for the Spatiotemporal Organization of Traction Forces during Cell Motility

Amoeboid motility results from pseudopod protrusions and retractions driven by traction forces of cells. We propose that the motor and actin-crosslinking functions of MyoII differentially control the temporal and spatial distribution of the traction forces, and establish mechanistic relationships between these distributions, enabling cells to move.

Roles of cell confluency and fluid shear in 3-dimensional intracellular forces in endothelial cells

We use a novel 3D inter-/intracellular force microscopy technique based on 3D traction force microscopy to measure the cell–cell junctional and intracellular tensions in subconfluent and confluent vascular endothelial cell (EC) monolayers under static and shear flow conditions. We found that z-direction cell–cell junctional tensions are higher in confluent EC monolayers than those in subconfluent ECs, which cannot be revealed in the previous 2D methods. Under static conditions, subconfluent cells are under spatially non-uniform tensions, whereas cells in confluent monolayers are under uniform tensions. The shear modulations of EC cytoskeletal remodeling, extracellular matrix (ECM) adhesions, and cell–cell junctions lead to significant changes in intracellular tensions. When a confluent monolayer is subjected to flow shear stresses with a high forward component comparable to that seen in the straight part of the arterial system, the intracellular and junction tensions preferentially increase along the flow direction over time, which may be related to the relocation of adherens junction proteins. The increases in intracellular tensions are shown to be a result of chemo-mechanical responses of the ECs under flow shear rather than a direct result of mechanical loading. In contrast, the intracellular tensions do not show a preferential orientation under oscillatory flow with a very low mean shear. These differences in the directionality and magnitude of intracellular tensions may modulate translation and transcription of ECs under different flow patterns, thus affecting their susceptibility for atherogenesis.

The Scar/WAVE complex is necessary for proper regulation of traction stresses during amoeboid motility

A combination of traction force and F-actin measurements shows that cells lacking either of the SCAR/WAVE complex proteins SCAR and PIR121 exhibit an altered cell motility cycle and spatiotemporal distribution of tractions stresses, which correlate in magnitude with F-actin levels.

An Oscillatory Contractile Pole-Force Component Dominates the Traction Forces Exerted by Migrating Amoeboid Cells

We used principal component analysis to dissect the mechanics of chemotaxis of amoeboid cells into a reduced set of dominant components of cellular traction forces and shape changes. The dominant traction force component in wild-type cells accounted for ~40% of the mechanical work performed by these cells, and consisted of the cell attaching at front and back contracting the substrate towards its centroid (pole-force). The time evolution of this pole-force component was responsible for the periodic variations of cell length and strain energy that the cells underwent during migration. We identified four additional canonical components, reproducible from cell to cell, overall accounting for an additional ~20% of mechanical work, and associated with events such as lateral protrusion of pseudopodia. We analyzed mutant strains with contractility defects to quantify the role that non-muscle Myosin II (MyoII) plays in amoeboid motility. In MyoII essential light chain null cells the polar-force component remained dominant. On the other hand, MyoII heavy chain null cells exhibited a different dominant traction force component, with a marked increase in lateral contractile forces, suggesting that cortical contractility and/or enhanced lateral adhesions are important for motility in this cell line. By compressing the mechanics of chemotaxing cells into a reduced set of temporally-resolved degrees of freedom, the present study may contribute to refined models of cell migration that incorporate cell-substrate interactions.

Distribution of traction forces associated with shape changes during amoeboid cell migration

Amoeboid motility results from the cyclic repetition of shape changes leading to periodic oscillations of the cell length (motility cycle). We analyze the dominant modes of shape change and their association to the traction forces exerted on the substrate using Principal Component Analysis (PCA) of time-lapse measurements of cell shape and traction forces in migrating Dictyostelium cells. Using wild-type cells (wt) as reference, we investigated Myosin II activity by studying Myosin II heavy chain null cells (mhcA-) and Myosin II essential light chain null cells (mlcE-). We found that wt, mlcE-and mhcA- cells utilize similar modes of shape changes during their motility cycle, although these shape changes are implemented at a slower pace in Myosin II null mutants. The number of dominant modes of shape changes is surprisingly few with only four modes accounting for 75% of the variance in all cases. The three principal shape modes are dilation/elongation, bending, and bulging of the front/back. The second mode, resulting from sideways protrusion/retraction, is associated to lateral asymmetries in the cell traction forces, and is significantly less important in mhcA- cells. These results indicate that the mechanical cycle of traction stresses and cell shape changes remains remarkably similar for all cell lines but is slowed down when myosin function is lost, probably due to a reduced control on the spatial organization of the traction stresses.

Three-Dimensional Traction Force Distribution in Migrating Amoeboid Cells

We have developed a method which enables us to determine cellular traction forces exerted perpendicular to the substrate in addition to the in-plane forces. This solution also enables to analyze the errors associated to existing two-dimensional traction cytometry methods, which assume either that the vertical displacements or that the vertical stresses are zero on the surface of the substrate. We obtain information about the substrate deformation by imaging a small volume of the elastic substrate with embedded fluorescent marker beads. Correlation with a reference image enables us to obtain the 3D deformation of the substrate. The corresponding traction forces are obtained by solving the elastostatic equation for a linearly elastic medium using the calculated deformation of the substrate. Our studies of Dictyostelium cells moving over flat substrates are designed to reveal the importance of various cytoskeletal components for the organization of the traction stresses in all three dimensions. We are looking at different Dictyostelium mutants with crosslinking defects, such as myosin II-null cells and cortexilin-null cells, in order to study the role that these crosslinkers play in the overall distribution of the traction forces. We find that wt Dictyostelium cells push on the substrate near the center of the cell and pull at the periphery. The magnitude of these perpendicular forces is comparable to the magnitude of the forces produced in the plane of the substrate. Our initial findings show that the effects of mutations on the parallel forces do not necessarily predict the effects on the perpendicular forces. For example, myosin II-null cells show a significant reduction of the front to back organization of the parallel traction forces while the push pull distribution of forces remains unaffected.

The Role of the Scar/WAVE Complex in the Mechanics of Cell Migration

Cell motility is integral to a wide spectrum of biological phenomena. It requires the spatiotemporal coordination of underlying biochemical processes, resulting in cyclic shape changes associated with mechanical events (the motility cycle). A major driving force of cell migration is the dendritic polymerization of actin at the leading edge, regulated through the nucleation activity of the Arp2/3 complex, activated by the Scar/WAVE complex. Our aim is to understand the effect of the different components of the Scar/WAVE complex in the mechanics and in particular the motility cycle of migrating cells.For this purpose, we acquired time-lapse recordings of cell shape and traction forces of Dictyostelium cells migrating on deformable substrates. We compared results for wild-type cells and cells lacking the Scar/WAVE complex proteins PIR121 (Sra-1/CYFIP/GEX-2) (pirA-) and SCAR (scrA-). We find that mutantcells move slower than wild-type, while maintaining the overall characteristics of the mechanical interaction with the substrate, attaching at front and back and contracting inwards. Although the distribution of applied forces is unchanged, their magnitude is lower than in wild-type for scrA- cells and higher for pirA- cells. This correlates with the F-actin content of the different cell lines corroborating a role for F-actin in determining the level of the traction stresses.In pirA- cells regularity of the motility cycle (quasiperiodic repetition of shape changes and strain energy deposited) seems to be reduced compared to wild-type. This suggests that proper regulation of the Scar/WAVE complex and its role in F-actin turnover is essential for amoeboid motility.

Spatiotemporal Analysis of Traction Work Produced by Migrating Amoeboid Cells

Amoeboid cell motility is a complicated process requiring the regulated activity and localization of many molecules and resulting in the cyclic repetition of a relatively small repertoire of shape changes. These changes are driven by the traction work produced by the cell, which can be estimated by measuring the forces and displacements exerted by the cells on their substrate during migration. We have developed and applied a novel implementation of Principal Component Analysis to identify and sort out the most important shape changes in terms of traction work produced by chemotaxing Dictyostelium cells. For this purpose, we acquired time-lapse recordings of cell shape and traction forces of Dictyostelium cells migrating on deformable substrates. Using wild-type cells as reference, we investigated the effect of altering myosin II activity by studying myosin II null cells and essential light chain null cells. Our results indicate that the spatio-temporal variation of the traction work produced by Dictyostelium cells can be described with a reduced number of modes. In fact, only four modes are needed to account for 65% of the traction work exerted by all cells lines studied. Furthermore, the first mode alone accounts for more than 40% of the traction work. Spatially, this mode consists of the attachment of the cell predominantly at two areas at front and back, contracting towards the center of the cell. The time evolution of this mode is approximately periodic and coincides with the time evolution of cell length. Each one of the remaining modes accounts for less that 10% of the traction work. Their temporal and spatial organization is less clear, suggesting that the cell performs a traction work cycle composed of a repetitive sequence of steps over which random fluctuations are imposed.