Free Form Deformation-Based Image Registration Improves Accuracy of Traction Force Microscopy

Traction Force Microscopy (TFM) is a widespread method used to recover cellular tractions from the deformation that they cause in their surrounding substrate. Particle Image Velocimetry (PIV) is commonly used to quantify the substrate's deformations, due to its simplicity and efficiency. However, PIV relies on a block-matching scheme that easily underestimates the deformations. This is especially relevant in the case of large, locally non-uniform deformations as those usually found in the vicinity of a cell's adhesions to the substrate. To overcome these limitations, we formulate the calculation of the deformation of the substrate in TFM as a non-rigid image registration process that warps the image of the unstressed material to match the image of the stressed one. In particular, we propose to use a B-spline -based Free Form Deformation (FFD) algorithm that uses a connected deformable mesh to model a wide range of flexible deformations caused by cellular tractions. Our FFD approach is validated in 3D fields using synthetic (simulated) data as well as with experimental data obtained using isolated endothelial cells lying on a deformable, polyacrylamide substrate. Our results show that FFD outperforms PIV providing a deformation field that allows a better recovery of the magnitude and orientation of tractions. Together, these results demonstrate the added value of the FFD algorithm for improving the accuracy of traction recovery.Funded by Ministerio de Economía y Competividad (ES); url: http://www.mineco.gob.es/; RyC2010-06094, Fundación Ramón Areces (ES); url: http://www.fundacionareces.es/fundacionareces/, Ministerío de Economía y Competividad (ES); url: http://www.mineco.gob.es/; SAF2011-24953 (MVM); Ministerio de Economía y Competividad (ES); url: http://www.mineco.gob.es/; DPI2012-38090-C1, European Research Council (BE); url: http://erc.europa.eu/; 306751 (JMGA); European Research Council (BE); url: http://erc.europa.eu/; 308223 (HVO); Ministerio de Economía y Competividad (ES); url: http://www.mineco.gob.es/; DPI2012-38090-C3 (COS); and Ministerio de Economía y Competividad (ES); url: http://www.mineco.gob.es/; TEC2013- 48552-C2-1-R (AMB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.European Community's Seventh Framework Progra

A regulatory motif in nonmuscle myosin II-B regulates its role in migratory front-back polarity

In this study, we show that the role of nonmuscle myosin II (NMII)-B in front–back migratory cell polarity is controlled by a short stretch of amino acids containing five serines (1935–1941). This motif resides near the junction between the C terminus helical and nonhelical tail domains. Removal of this motif inhibited NMII-B assembly, whereas its insertion into NMII-A endowed an NMII-B–like ability to generate large actomyosin bundles that determine the rear of the cell. Phosphomimetic mutation of the five serines also inhibited NMII-B assembly, rendering it unable to support front–back polarization. Mass spectrometric analysis showed that several of these serines are phosphorylated in live cells. Single-site mutagenesis showed that serine 1935 is a major regulatory site of NMII-B function. These data reveal a novel regulatory mechanism of NMII in polarized migrating cells by identifying a key molecular determinant that confers NMII isoform functional specificityThis work is supported by grants SAF2011-24953 from MINECO, FP7 Marie Curie CIG-293719 from the EU, CIVP16A1831 from the Ramon Areces Foundation (M. Vicente-Manzanares), GM 23244 (A.R. Horwitz), GM037537 (D.F. Hunt), and the Cell Migration Consortium U54 GM64346 (A.R. Horwitz and D.F. Hunt). M. Vicente-Manzanares is an investigator from the Ramón y Cajal Program (RYC-2010-06094)

Multiple-level su-8 µtas chip transfer onto coverslips for biological applications

Trabajo presentado en MicroTAS 2021, celebrado en modalidad virtual del 10 al 14 de octubre de 2021.In this work, we present a chip-on-a-coverslip consisting of customized multiple-layer SU-8 structures transferred onto standard glass coverslips for their direct application in confocal microscopy. This approach overcomes inherent challenges with photolithography over small substrates. We demonstrate their functionality with bespoke durotaxis chips to observe the interaction between cells and buried mechanical cues

Full L-1-regularized Traction Force Microscopy over whole cells

Traction Force Microscopy (TFM) is a widespread technique to estimate the tractions that cells exert on the surrounding substrate. To recover the tractions, it is necessary to solve an inverse problem, which is ill-posed and needs regularization to make the solution stable. The typical regularization scheme is given by the minimization of a cost functional, which is divided in two terms: the error present in the data or data fidelity term; and the regularization or penalty term. The classical approach is to use zero-order Tikhonov or L2-regularization, which uses the L2-norm for both terms in the cost function. Recently, some studies have demonstrated an improved performance using L1-regularization (L1-norm in the penalty term) related to an increase in the spatial resolution and sensitivity of the recovered traction field. In this manuscript, we present a comparison between the previous two regularization schemes (relying in the L2-norm for the data fidelity term) and the full L1-regularization (using the L1-norm for both terms in the cost function) for synthetic and real data. Our results reveal that L1-regularizations give an improved spatial resolution (more important for full L1-regularization) and a reduction in the background noise with respect to the classical zero-order Tikhonov regularization. In addition, we present an approximation, which makes feasible the recovery of cellular tractions over whole cells on typical full-size microscope images when working in the spatial domain. The proposed full L1-regularization improves the sensitivity to recover small stress footprints. Moreover, the proposed method has been validated to work on full-field microscopy images of real cells, what certainly demonstrates it is a promising tool for biological applications.status: publishe

A regulatory motif in nonmuscle myosin II-B regulates its role in migratory front-back polarity

In this study, we show that the role of nonmuscle myosin II (NMII)-B in front-back migratory cell polarity is controlled by a short stretch of amino acids containing five serines (1935-1941). This motif resides near the junction between the C terminus helical and nonhelical tail domains. Removal of this motif inhibited NMII-B assembly, whereas its insertion into NMII-A endowed an NMII-B-like ability to generate large actomyosin bundles that determine the rear of the cell. Phosphomimetic mutation of the five serines also inhibited NMII-B assembly, rendering it unable to support front-back polarization. Mass spectrometric analysis showed that several of these serines are phosphorylated in live cells. Single-site mutagenesis showed that serine 1935 is a major regulatory site of NMII-B function. These data reveal a novel regulatory mechanism of NMII in polarized migrating cells by identifying a key molecular determinant that confers NMII isoform functional specificity.This work is supported by grants SAF2011-24953 from MINECO, FP7 Marie Curie CIG-293719 from the EU, CIVP16A1831 from the Ramon Areces Foundation (M. Vicente-Manzanares), GM 23244 (A.R. Horwitz), GM037537 (D.F. Hunt), and the Cell Migration Consortium U54 GM64346 (A.R. Horwitz and D.F. Hunt). M. Vicente-Manzanares is an investigator from the Ramón y Cajal Program (RYC-2010-06094).Peer Reviewe

Free Form Deformation -based Image Registration Improves Accuracy of Traction Force Microscopy

Traction Force Microscopy (TFM) is a widespread method used to recover cellular tractions from the deformation that they cause in their surrounding substrate. Particle Image Velocimetry (PIV) is commonly used to quantify the substrate’s deformations, due to its simplicity and efficiency. However, PIV relies on a block-matching scheme that easily underestimates the deformations. This is especially relevant in the case of large, locally non-uniform deformations as those usually found in the vicinity of a cell’s adhesions to the substrate. To overcome these limitations, we formulate the calculation of the deformation of the substrate in TFM as a non-rigid image registration process that warps the image of the unstressed material to match the image of the stressed one. In particular, we propose to use a B-spline -based Free Form Deformation (FFD) algorithm that uses a connected deformable mesh to model a wide range of flexible deformations caused by cellular tractions. Our FFD approach is validated in 3D fields using synthetic (simulated) data as well as with experimental data obtained using isolated endothelial cells lying on a deformable, polyacrylamide substrate. Our results show that FFD outperforms PIV providing a deformation field that allows a better recovery of the magnitude and orientation of tractions. Together, these results demonstrate the added value of the FFD algorithm for improving the accuracy of traction recovery.status: publishe

Tyrosine phosphorylation of the myosin regulatory light chain controls non-muscle myosin II assembly and function in migrating cells

Active non-muscle myosin II (NMII) enables migratory cell polarization and controls dynamic cellular processes, such as focal adhesion formation and turnover and cell division. Filament assembly and force generation depend on NMII activation through the phosphorylation of Ser19 of the regulatory light chain (RLC). Here, we identify amino acid Tyr (Y) 155 of the RLC as a novel regulatory site that spatially controls NMII function. We show that Y155 is phosphorylated in vitro by the Tyr kinase domain of epidermal growth factor (EGF) receptor. In cells, phosphorylation of Y155, or its phospho-mimetic mutation (Glu), prevents the interaction of RLC with the myosin heavy chain (MHCII) to form functional NMII units. Conversely, Y155 mutation to a structurally similar but non-phosphorylatable amino acid (Phe) restores the more dynamic cellular functions of NMII, such as myosin filament formation and nascent adhesion assembly, but not those requiring stable actomyosin bundles, e.g., focal adhesion elongation or migratory front-back polarization. In live cells, phospho-Y155 RLC is prominently featured in protrusions, where it prevents NMII assembly. Our data indicate that Y155 phosphorylation constitutes a novel regulatory mechanism that contributes to the compartmentalization of NMII assembly and function in live cells.C.L.-G. is supported by a predoctoral fellowship from the Junta de Castilla y León. M.M.-S. is supported by a predoctoral fellowhip from the AECC. This work was funded by the following grants: Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia (CLC–2017–01) from the Junta de Castilla-León with FEDER funds (Spain); SAF2014-54705-R and SAF2017-87408-R from MINECO (Spain); CIVP16A1831 from the Ramón Areces Foundation (Spain); 14-BBM-340 from the BBVA Foundation (Spain); and IDEAS-VICE18 from the Asociacion Española Contra el Cáncer (AECC, Spain) to M.V.-M.; NIH GM 037537 (D.F.H.); and K22HL131869 (S.M.H.) and the Intramural Research Program (J.R.S.) of the National Heart, Lung, and Blood Institute, NIH.Peer reviewe

Displacements obtained from a circular traction patch of 6μm diameter exerting a load of 10% of the Young modulus along the X-axis.

<p>(a) Magnitude of the displacement field provided by the simulated ground-truth data, (b) PIV algorithm and (c) FFD algorithm. Cones indicate the direction of the field at those locations where the magnitude is larger than 20% of the peak magnitude. Units are given in μm. The scale bars represent 5μm.</p

Tractions obtained from a circular traction patch of 6μm diameter exerting a load of 10% of the Young modulus along the (negative) Z-axis.

<p>From left to right, simulated tractions, results from PIV, and results from FFD. (a) Magnitude of the traction field before and after segmenting the stress footprint and, (b) its corresponding orientation with the elevation angle indicated by the colormap. Units of the magnitude are given as percentage of the Young modulus. Units of the elevation angles are given in degrees (-90 corresponding to negative Z-axis). The scale bar represents 5μm.</p

Recovered stress footprints.

<p>Recovered stress footprints for tractions with magnitudes of 15%, 10% and 5% of the substrate Young’s modulus, aligned with the X and Z Cartesian directions, and distributed over a circular area of 10μm, 6μm and 4μm diameter. Units of color bars are given as percentage of the Young’s modulus.</p