Publications

Deep Learning (DL) algorithms have been used to model physical systems. However, the success heavily relies on how DL methodologies accommodate the properties of a system and data. (…) Under this context, we study the case of Traction Force Microscopy (TFM), a class of experimental procedures and algorithms for measuring cell traction. (…) We introduce Deep Morphology Traction Microscopy (DeepMorphoTM), a DL approach that infers cell traction from a shape sequence of a cell. By employing a deterministic framework, DeepMorphoTM effectively mitigates the biological variability in cell contractility for a given cell shape. …

The physical structure and dynamics of cells are supported by micron-scale actin networks with diverse geometries, protein compositions, and mechanical properties. These networks are composed of actin filaments and numerous actin binding proteins (ABPs), many of which engage multiple filaments simultaneously to crosslink them into specific functional architectures. Mechanical force has been shown to modulate the interactions between several ABPs and individual actin filaments, but it is unclear how this phenomenon contributes to the emergent force-responsive functional dynamics of actin networks. Here, we engineer filament linker complexes and combine them with photo-micropatterning of myosin motor proteins to produce an in vitro reconstitution platform for examining how force impacts the behavior of ABPs within multi-filament assemblies. …

Naïve T lymphocytes traffic through the organism in search for antigen, alternating between blood and secondary lymphoid organs. Lymphocyte homing to lymph nodes relies on CCL21 chemokine sensing by CCR7 receptors, while exit into efferent lymphatics relies on sphingolipid S1P sensing by S1PR1 receptors. While both molecules are claimed chemotactic, a quantitative analysis of naïve T lymphocyte migration along defined gradients is missing. Here, we used a reductionist approach to study the real-time single-cell response of naïve T lymphocytes to CCL21 and serum rich in bioactive S1P. Using microfluidic and micropatterning ad hoc tools, we show that CCL21 triggers stable polarization and long-range chemotaxis of cells, whereas S1P-rich serum triggers a transient polarization only and no significant displacement, potentially representing a brief transmigration step through exit portals. …

Cryogenic-electron tomography (cryo-ET) has provided an unprecedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare adherent cell types for cryo-ET as it promotes optimal positioning of cells and subcellular regions of interest for vitrification, cryo-focused ion beam (cryo-FIB) milling, and data acquisition. Here we demonstrate a micropatterning workflow for capturing minimally adherent cell types, human T-cells and Jurkat cells, for cryo-FIB and cryo-ET. …

Blood platelets are central elements of the blood clotting response after wounding. Upon vessel damage, they bind to the surrounding matrix and contract the forming thrombus, thus helping to restore normal blood circulation. The hemostatic function of platelets is directly connected to their mechanics and cytoskeletal organization. The reorganization of the platelet cytoskeleton during spreading occurs within minutes and leads to the formation of contractile actomyosin bundles, but it is not known if there is a direct correlation between the emerging actin structures and the force field that is exerted to the environment. In this study, we combine fluorescence imaging of the actin structures with simultaneous traction force measurements in a time-resolved manner. …

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. …

The RAC1-WAVE-Arp2/3 signaling pathway generates branched actin networks that power lamellipodium protrusion of migrating cells. Feedback is thought to control protrusion lifetime and migration persistence, but its molecular circuitry remains elusive. Here, we identify PPP2R1A by proteomics as a protein differentially associated with the WAVE complex subunit ABI1 when RAC1 is activated and downstream generation of branched actin is blocked. …

Spatial patterns of adhesive substrates can dictate cell motility trajectories.
Mammalian cells demonstrate idiosyncratic trajectories when subjected to barbell-shaped micropatterns with thin constrictions. However, the morphologies of these cells over time remained uncharacterized. We subjected 3T3 mouse fibroblast cells to an array of micropatterns, and compared their shape dynamics to a panel of triple-negative breast cancer cells. When exposed to a gradient of size, we found that the morphological dynamics of 3T3 cells display trends analogous to the potential energy curve of a molecule, where energy is high at small micropattern sizes, drops off at a minimum point, and rises again at large micropattern sizes. We also
found the breast cancer cell panel exhibited a gradient of morphological activity and could be classified using principal component analysis. …

Notochord progenitors (NotoPs) are a rare, yet vital embryonic cell population that give rise to the cells that form and maintain intervertebral discs. An unlimited access to NotoPs would open new opportunities for basic biomedical research and regenerative medicine of the discs. However, the mechanisms responsible for the specification and the maintenance of NotoPs are not understood. This gap in understanding stems from the fact that NotoPs emerge during the gastrulation to axial elongation transition; an event that is ethically and technically challenging to investigate. Here, to circumvent this issue, we use micropatterning to guide the development of human ESCs into standardised patterns of anterior primitive streak cell fates. …

Platforms with nanoscale topography have recently become powerful tools in cellular biophysics and bioengineering. Recent studies have shown that nanotopography affects various cellular processes like adhesion and endocytosis, as well as physical properties such as cell shape. To engineer nanopillars more effectively for biomedical applications, it is crucial to gain better control and understanding of how nanopillars affect cell and nuclear physical properties, such as shape and spreading area, and impact cellular processes like endocytosis and adhesion. In this study, we utilized a laser-assisted micropatterning technique to manipulate the 2D architectures of cells on 3D nanopillar platforms. We performed a comprehensive analysis of cellular and nuclear morphology and deformation on both nanopillar and flat substrates. …

Realistic models of the human vasculature would benefit understanding of normal physiology and disease pathology in the blood circulatory systems. Here we used a photo patterning system to form near vertical hydrogel walls inside a microfluidic device, generating a perfusable network. The hydrogels had realistic (tissue-like) viscoelastic properties and were permeable to large molecules like 70kD dextran. Endothelial cells from human pluripotent stem cells (hiPSC-ECs) cultured in the hydrogels could be guided to form complex networks. …

Actin cytoskeleton contractility plays a critical role in morphogenetic processes by generating forces that are then transmitted to cell–cell and cell-ECM adhesion complexes. In turn, mechanical properties of the environment are sensed and transmitted to the cytoskeleton at cell adhesion sites, influencing cellular processes such as cell migration, differentiation and survival. (…) A key issue is to be able to measure the forces generated by actomyosin and transmitted to the adhesion complexes. Here, we applied an intermediate approach allowing reconstruction of the actomyosin-α-catenin complex in acellular conditions to probe directly the transmitted forces. For this, we combined micropatterning of purified α-catenin and spontaneous actomyosin network assembly in the presence of G-actin and Myosin II with micro force sensor arrays used so far to measure cell-generated forces. …

Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. Our data define a central role of B-plexins in mechanosensation to couple cell density and cell division in development and disease. We identify a critical requirement of B-plexin transmembrane receptors in the response to crowding-induced mechanical forces during embryonic skin development. We show that B-plexins mediate mechanoresponses to crowding through stabilization of adhesive cell junctions and lowering of cortical stiffness; and provide evidence that the B-plexin-dependent mechanochemical feedback is also pathophysiologically relevant to limit tumor growth in basal cell carcinoma, the most common type of skin cancer. …

Jimenez et al. show that the centrosome, which has long been thought to sit at the geometric center of the cell, is actually positioned at the center of a subcellular zone defined by the absence of contractile acto-myosin bundles. Centrosome position is defined by dyneins exerting pulling forces on microtubules specifically in this zone. …

During embryonic and postnatal development, or- gans and tissues grow steadily to achieve their final size at the end of puberty. However, little is known about the cellular dynamics that mediate postnatal growth. By combining in vivo clonal lineage tracing, proliferation kinetics, single-cell transcriptomics, and in vitro micro-pattern experiments, we resolved the cellular dynamics taking place during postnatal skin epidermis expansion. Our data revealed that harmonious growth is engineered by a single popula- tion of developmental progenitors presenting…