Adithan Kandasamy

All Publications

• Uncertainty-aware traction force microscopy. PLoS computational biology Kandasamy, A., Yeh, Y. T., Serrano, R., Mercola, M., Del Alamo, J. C. 2025; 21 (6): e1013079

  Abstract

  Traction Force Microscopy (TFM) is a versatile tool to quantify cell-exerted forces by imaging and tracking fiduciary markers embedded in elastic substrates. The computations involved in TFM are often ill-conditioned, and data smoothing or regularization is required to avoid overfitting the noise in the tracked displacements. Most TFM calculations depend critically on the heuristic selection of regularization (hyper-) parameters affecting the balance between overfitting and smoothing. However, TFM methods rarely estimate or account for measurement errors in substrate deformation to adjust the regularization level accordingly. Moreover, there is a lack of tools for uncertainty quantification (UQ) to understand how these errors propagate to the recovered traction stresses. These limitations make it difficult to interpret the TFM readouts and hinder comparing different experiments. This manuscript presents an uncertainty-aware TFM technique that estimates the variability in the magnitude and direction of the traction stress vector recovered at each point in space and time of each experiment. In this technique, a non-parametric bootstrap method perturbs the cross-correlation functional of Particle Image Velocimetry (PIV) to assess the uncertainty of the measured deformation. This information is passed on to a hierarchical Bayesian TFM framework with spatially adaptive regularization that propagates the uncertainty to the traction stress readouts (TFM-UQ). We evaluate TFM-UQ using synthetic datasets with prescribed image quality variations and demonstrate its application to experimental datasets. These studies show that TFM-UQ bypasses the need for subjective regularization parameter selection and locally adapts smoothing, outperforming traditional regularization methods. They also illustrate how uncertainty-aware TFM tools can be used to objectively choose key image analysis parameters like PIV window size. We anticipate that these tools will allow for decoupling biological heterogeneity from measurement variability and facilitate automating the analysis of large datasets by parameter-free, input data-based regularization.

  View details for DOI 10.1371/journal.pcbi.1013079

  View details for PubMedID 40505016

• Tunable photoinitiated hydrogel microspheres for quantifying cell-generated forces in complex three-dimensional environments. Acta biomaterialia Garcia-Herreros, A., Yeh, Y. T., Tu, Y., Kandasamy, A., Del Alamo, J. C., Criado-Hidalgo, E. 2025; 205: 521-536

  Abstract

  We present a high-throughput method using standard laboratory equipment and microfluidics to produce cellular force microscopy probes with controlled size and elastic modulus. Mechanical forces play crucial roles in cell biology but quantifying these forces in physiologically relevant systems remains challenging due to the complexity of the native cell environment. Polymerized hydrogel microspheres offer great promise for interrogating the mechanics of processes inaccessible to classic force microscopy methods. However, despite significant recent advances, their small size and large surface-to-volume ratio impede the high-yield production of probes with tunable, monodisperse distributions of size and mechanical properties. To overcome these limitations, we use a flow-focusing microfluidic device to generate large quantities of droplets with highly reproducible, adjustable radii. These droplets contain acrylamide gel precursor and the photoinitiator Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a source of free radicals. LAP provides fine control over microsphere polymerization due to its high molar absorptivity at UV wavelengths and moderate water solubility. The polymerized microspheres can be functionalized with different conjugated extracellular matrix proteins and embedded with fluorescent nanobeads to promote cell attachment and track microsphere deformation. As proof of concept, we measure the mechanical forces generated by a monolayer of vascular endothelial cells engulfing functionalized microspheres. Individual nanobead motions are tracked and analyzed to determine 3D traction forces via direct computation of stress from measured strain. These results reveal that the cell monolayer collectively exerts strong radial compression on the encapsulated probe, suggesting new biomechanical functions of endothelial cells that could modulate diapedesis or pathogen internalization. STATEMENT OF SIGNIFICANCE: Mechanical forces are crucial to many cell biology processes but quantifying them in complex native environments remains challenging. We address this by introducing linearly elastic probes with known mechanical properties, whose deformations can be accurately measured to infer local stresses. Specifically, we present a high-throughput method for producing polyacrylamide (PAAm) hydrogel microspheres embedded with fluorescent nanoparticles. To measure cell-generated forces in physiologically relevant systems, the probes are tracked using a 3D coherent point drift algorithm, yielding high-resolution deformation data with minimal computational cost. This method overcomes key barriers in PAAm microsphere fabrication by ensuring monodisperse size, tunable stiffness, and simple, reproducible processes suitable for most cell biology labs-making it a powerful tool for studying cellular mechanobiology.

  View details for DOI 10.1016/j.actbio.2025.08.041

  View details for PubMedID 40882905

• FMNL1 and mDia1 promote efficient T cell migration through complex environments via distinct mechanisms. Frontiers in immunology Sigler, A. L., Thompson, S. B., Ellwood-Digel, L., Kandasamy, A., Michaels, M. J., Thumkeo, D., Narumiya, S., Del Alamo, J. C., Jacobelli, J. 2024; 15: 1467415

  Abstract

  Lymphocyte trafficking and migration through tissues is critical for adaptive immune function and, to perform their roles, T cells must be able to navigate through diverse tissue environments that present a range of mechanical challenges. T cells predominantly express two members of the formin family of actin effectors, Formin-like 1 (FMNL1) and mammalian diaphanous-related formin 1 (mDia1). While both FMNL1 and mDia1 have been studied individually, they have not been directly compared to determine functional differences in promoting T cell migration. Through in vivo analysis and the use of in vitro 2D and 3D model environments, we demonstrate that FMNL1 and mDia1 are both required for effective T cell migration, but they have different localization and roles in T cells, with specific environment-dependent functions. We found that mDia1 promotes general motility in 3D environments in conjunction with Myosin-II activity. We also show that, while mDia1 is almost entirely in the cytoplasmic compartment, a portion of FMNL1 physically associates with the nucleus. Furthermore, FMNL1 localizes to the rear of migrating T cells and contributes to efficient migration by promoting deformation of the rigid T cell nucleus in confined environments. Overall, our data indicates that while FMNL1 and mDia1 have similar mechanisms of actin polymerization, they have distinct roles in promoting T cell migration. This suggests that differential modulation of FMNL1 and mDia1 can be an attractive therapeutic route to fine-tune T cell migration behavior.

  View details for DOI 10.3389/fimmu.2024.1467415

  View details for PubMedID 39430739

  View details for PubMedCentralID PMC11486666

• Open-source algorithm for high-throughput, non-contact elasticity measurements of IPSC-derived cardiomyocytes Kandasamy, A., Serrano, R., Mercola, M., Del Alamo, J. CELL PRESS. 2024: 403A

  View details for Web of Science ID 001194120702359

• Distinct platelet F-actin patterns and traction forces on von Willebrand factor versus fibrinogen. Biophysical journal Mollica, M. Y., Beussman, K. M., Kandasamy, A., Rodríguez, L. M., Morales, F. R., Chen, J., Manohar, K., Del Álamo, J. C., López, J. A., Thomas, W. E., Sniadecki, N. J. 2023; 122 (18): 3738-3748

  Abstract

  Upon vascular injury, platelets form a hemostatic plug by binding to the subendothelium and to each other. Platelet-to-matrix binding is initially mediated by von Willebrand factor (VWF) and platelet-to-platelet binding is mediated mainly by fibrinogen and VWF. After binding, the actin cytoskeleton of a platelet drives its contraction, generating traction forces that are important to the cessation of bleeding. Our understanding of the relationship between adhesive environment, F-actin morphology, and traction forces is limited. Here, we examined F-actin morphology of platelets attached to surfaces coated with fibrinogen and VWF. We identified distinct F-actin patterns induced by these protein coatings and found that these patterns were identifiable into three classifications via machine learning: solid, nodular, and hollow. We observed that traction forces for platelets were significantly higher on VWF than on fibrinogen coatings and these forces varied by F-actin pattern. In addition, we analyzed the F-actin orientation in platelets and noted that their filaments were more circumferential when on fibrinogen coatings and having a hollow F-actin pattern, while they were more radial on VWF and having a solid F-actin pattern. Finally, we noted that subcellular localization of traction forces corresponded to protein coating and F-actin pattern: VWF-bound, solid platelets had higher forces at their central region while fibrinogen-bound, hollow platelets had higher forces at their periphery. These distinct F-actin patterns on fibrinogen and VWF and their differences in F-actin orientation, force magnitude, and force localization could have implications in hemostasis, thrombus architecture, and venous versus arterial thrombosis.

  View details for DOI 10.1016/j.bpj.2023.07.006

  View details for PubMedID 37434354

  View details for PubMedCentralID PMC10541491

• Biomechanical interactions of Schistosoma mansoni eggs with vascular endothelial cells facilitate egg extravasation. PLoS pathogens Yeh, Y. T., Skinner, D. E., Criado-Hidalgo, E., Chen, N. S., Garcia-De Herreros, A., El-Sakkary, N., Liu, L., Zhang, S., Kandasamy, A., Chien, S., Lasheras, J. C., Del Álamo, J. C., Caffrey, C. R. 2022; 18 (3): e1010309

  Abstract

  The eggs of the parasitic blood fluke, Schistosoma, are the main drivers of the chronic pathologies associated with schistosomiasis, a disease of poverty afflicting approximately 220 million people worldwide. Eggs laid by Schistosoma mansoni in the bloodstream of the host are encapsulated by vascular endothelial cells (VECs), the first step in the migration of the egg from the blood stream into the lumen of the gut and eventual exit from the body. The biomechanics associated with encapsulation and extravasation of the egg are poorly understood. We demonstrate that S. mansoni eggs induce VECs to form two types of membrane extensions during encapsulation; filopodia that probe eggshell surfaces and intercellular nanotubes that presumably facilitate VEC communication. Encapsulation efficiency, the number of filopodia and intercellular nanotubes, and the length of these structures depend on the egg's vitality and, to a lesser degree, its maturation state. During encapsulation, live eggs induce VEC contractility and membranous structures formation in a Rho/ROCK pathway-dependent manner. Using elastic hydrogels embedded with fluorescent microbeads as substrates to culture VECs, live eggs induce VECs to exert significantly greater contractile forces during encapsulation than dead eggs, which leads to 3D deformations on both the VEC monolayer and the flexible substrate underneath. These significant mechanical deformations cause the VEC monolayer tension to fluctuate with the eventual rupture of VEC junctions, thus facilitating egg transit out of the blood vessel. Overall, our data on the mechanical interplay between host VECs and the schistosome egg improve our understanding of how this parasite manipulates its immediate environment to maintain disease transmission.

  View details for DOI 10.1371/journal.ppat.1010309

  View details for PubMedID 35316298

  View details for PubMedCentralID PMC8939816

• The interplay between matrix deformation and the coordination of turning events governs directed neutrophil migration in 3D matrices. Science advances François, J., Kandasamy, A., Yeh, Y. T., Schwartz, A., Ayala, C., Meili, R., Chien, S., Lasheras, J. C., Del Álamo, J. C. 2021; 7 (29)

  Abstract

  Neutrophils migrating through extravascular spaces must negotiate narrow matrix pores without losing directional movement. We investigated how chemotaxing neutrophils probe matrices and adjust their migration to collagen concentration ([col]) changes by tracking 20,000 cell trajectories and quantifying cell-generated 3D matrix deformations. In low-[col] matrices, neutrophils exerted large deformations and followed straight trajectories. As [col] increased, matrix deformations decreased, and neutrophils turned often to circumvent rather than remodel matrix pores. Inhibiting protrusive or contractile forces shifted this transition to lower [col], implying that mechanics play a crucial role in defining migratory strategies. To balance frequent turning and directional bias, neutrophils used matrix obstacles as pivoting points to steer toward the chemoattractant. The Actin Related Protein 2/3 complex coordinated successive turns, thus controlling deviations from chemotactic paths. These results offer an improved understanding of the mechanisms and molecular regulators used by neutrophils during chemotaxis in restrictive 3D environments.

  View details for DOI 10.1126/sciadv.abf3882

  View details for PubMedID 34261650

  View details for PubMedCentralID PMC8279509