During chemotactic movement, D. discoideum exhibits step-wise amoeboid motility driven by both contractile axial forces and lateral forces.

Leukocyte transmigration across vessel walls is a critical step in the innate immune response. Upon their activation and firm adhesion to vascular endothelial cells (VECs), leukocytes preferentially extravasate across junctional gaps in the endothelial monolayer (paracellular diapedesis). It has been hypothesized that VECs facilitate paracellular diapedesis by opening their cell-cell junctions in response to the presence of an adhering leukocyte. However, it is unclear how leukocytes interact mechanically with VECs to open the VEC junctions and migrate across the endothelium. In this study, we measured the spatial and temporal evolution of the 3D traction stresses generated by the leukocytes and VECs to elucidate the sequence of mechanical events involved in paracellular diapedesis. Our measurements suggest that the contractile stresses exerted by the leukocytes and the VECs can separately perturb the junctional tensions of VECs to result in the opening of gaps before the initiation of leukocyte transmigration. Decoupling the stresses exerted by the transmigrating leukocytes and the VECs reveals that the leukocytes actively contract the VECs to open a junctional gap and then push themselves across the gap by generating strong stresses that push into the matrix. In addition, we found that diapedesis is facilitated when the tension fluctuations in the VEC monolayer were increased by proinflammatory thrombin treatment. Our findings demonstrate that diapedesis can be mechanically regulated by the transmigrating leukocytes and by proinflammatory signals that increase VEC contractility.

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.

The mechanical environment of a cell influences its migration. It has been observed that certain cell types such as Dictyostelium and cancer cells tend to migrate by forming blebs rather than pseudopodia in mechanically resistive environment. However, very little is known about the mechanisms governing this transition between different modes of migration. In this work, we disentangle the contributions of two key mechanical factors that might trigger this switch, the environment's stiffness (the extent of local deformation of extra-cellular matrix) and its state of stress (amount of pre-existing tension or compression).

Three-dimensional (3-D) neutrophil migration is essential for immune surveillance and inflammatory responses. During 3-D migration, especially through extravascular spaces, neutrophils rely on frontal protrusions and rear contractions to squeeze and maneuver through extracellular matrices containing narrow pores. However, the role of matrix density and the cells’ ability to probe and remodel matrix pores during 3-D chemotaxis are far from being understood. We investigated these processes by tracking the trajectories of over 20,000 neutrophils in a 3-D migration device containing collagen matrices of varying concentrations and analyzing the shape of these trajectories at multiple scales. Additionally, we quantified the transient 3-D matrix deformations caused by the migrating cells. The mean pore size of our reconstituted collagen matrices decreased when the collagen concentration ([col]) was increased. In low-[col] matrices, neutrophils exerted large transient deformations and migrated in relatively straight trajectories. In contrast, they were not able to appreciably deform high- [col] matrices and adapted to this inability by turning more often to circumvent these narrow matrix pores. While this adaptation resulted in slower migration, the cells were able to balance the more frequent turning with the long-range directional bias necessary for chemotaxis. Based on our statistical analysis of cell trajectories, we postulate that neutrophils achieve this balance by using matrix obstacles as pivoting points to steer their motion towards the chemoattractant. Inhibiting myosin-II contractility or Arp2/3-mediated pseudopod protrusions not only compromised the cells’ ability to deform the matrix, but also made them switch to increased turning in more restrictive matrices when compared to untreated control cells. Both myosin-II contractility and Arp2/3-mediated branched polymerization of actin played a role in fast migration, but Arp2/3 was also crucial for neutrophils when coordinating the orientations of successive turns to prevent veering away from the chemotactic path. These results may contribute to an improved understanding of the mechanisms employed by migrating neutrophils in confined 3-D environments, as well as the molecular and environmental regulators for maintaining persistent motion.

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Neutrophils are vital for inflammation and immune defense. Dependent on β2 integrins, spherical neutrophils spread on vascular endothelium after arrest, which is critical for their recruitment from circulation to resist high shear flow and to initiate intravascular crawling. Here, we use high‐resolution quantitative dynamic footprinting microscopy to monitor neutrophil spreading on a substrate of recombinant ICAM‐1 and P‐selectin under flow. A homogenous binding assay using the conformation‐reporter antibodies mAb24 (reporting high‐affinity β2, H+) and KIM127 (reporting extended β2, E+) showed three conformations of activated β2 integrins. E−H+ β2 integrins increased before E+H− and E+H+ conformations at the beginning of neutrophil spreading. Integrin extension depended on Syk‐mediated integrin outside‐in signaling. The ring of E−H+ and E+H+, but not E+H− β2 integrins was fully formed during late neutrophil spreading just before migration. Using kindlin‐3‐GFP fusion proteins, a ring of kindlin‐3 was observed before the ring of H+ integrins appeared. These findings show spatially coordinated integrin activation during spreading. The previously unrecognized E−H+ conformation is the pioneer integrin for neutrophil spreading and appears to be organized by kindlin‐3. Support or Funding Information This research was supported by funding from the National Institutes of Health, USA (NIH, HL078784 and R01HL145454), WSA postdoctoral fellowships and a Career Development Award from the American Heart Association, USA (AHA, 16POST31160014, 19POST34450228, and 18CDA34110426). Integrin activation on a spread neutrophil footprint. Red is the mAb KIM127, which reports integrin extension; Green is the mAb24, which reports integrin high‐affinity; Blue is conformational non‐specific aLb2 integrins. Kindlin‐3‐GFP molecules formed a ring after the spreading of a neutrophil‐like HL60 cell.

B cells are activated by the binding of membrane-bound antigen to the B cell receptor (BCR), which induces actin dynamics, reorganization of receptors into signaling microclusters, and cell spreading. In vivo, B cells gather antigen from a variety of sources which may have different physical characteristics such as mobility, stiffness or topography. However, the effect of these physical parameters on BCR clustering and signaling activation is not understood.Here we have studied the role of topography of the stimulating surface on cell spreading, actin polymerization and signaling activation. BCR ligand coated substrates presenting ridges of variable spacing were used to probe the interaction of B cells with non-planar surfaces. Using high-resolution TIRF and confocal microscopy of live cells, we followed the movement of BCR clusters and actin dynamics. We found that small ridge separations induced actin waves that travel parallel to the ridges, resulting in protrusions and retractions of the cell edge. Large ridge separations result in global oscillations of actin intensity in the vicinity of ridges. We further investigated the temporal dynamics of calcium enrichment after antigen engagement in B cells. On flat substrates we measured periodic oscillations of calcium influx with a period of about 30 s, consistent with previously observed values. Interestingly, we found the period of calcium enrichment was dependent on ridge spacing, with increasing time intervals on smaller spacings. Drugs that inhibited actin dynamics slowed down the observed oscillations of calcium. Our results indicate that B cells are sensitive to topographical features, resulting in modulated actin dynamics and that calcium signaling is coupled to substrate-proximal actin dynamics.