Mammalian cells developed two main migration modes. The slow mesenchymatous mode, like crawling of fibroblasts, relies on maturation of adhesion complexes and actin fibre traction, while the fast amoeboid mode, observed exclusively for leukocytes and cancer cells, is characterized by weak adhesion, highly dynamic cell shapes, and ubiquitous motility on 2D and in 3D solid matrix. In both cases, interactions with the substrate by adhesion or friction are widely accepted as a prerequisite for mammalian cell motility, which precludes swimming. We show here experimental and computational evidence that leukocytes do swim, and that efficient propulsion is not fuelled by waves of cell deformation but by a rearward and inhomogeneous treadmilling of the cell external membrane. Our model consists of a molecular paddling by transmembrane proteins linked to and advected by the actin cortex, whereas freely diffusing transmembrane proteins hinder swimming. Furthermore, continuous paddling is enabled by a combination of external treadmilling and selective recycling by internal vesicular transport of cortex-bound transmembrane proteins. This mechanism explains observations that swimming is five times slower than the retrograde flow of cortex, and also that lymphocytes are motile in non-adherent confined environments. Resultantly, the ubiquitous ability of mammalian amoeboid cells to migrate in 2D or 3D, and with or without adhesion, can be explained for lymphocytes by a single machinery of heterogeneous membrane treadmilling. SIGNIFICANCE STATEMENT Leukocytes have a ubiquitous capacity to migrate on or in solid matrices, and with or without adhesion, which is instrumental to fight infections. The precise mechanisms sustaining migration remain however arguable. It is for instance widely accepted that leukocytes cannot crawl on 2D substrates without adhesion. In contrast, we showed that human lymphocytes swim on nonadherent 2D substrates and in suspension. Furthermore, our experiments and modelling suggest that propulsion rely hardly on cell body deformations and predominantly on molecular paddling by transmembrane proteins protruding outside the cell. For physics, this study reveals a new type of micro-swimmer, and for biology it suggests that leukocytes ubiquitous crawling may have evolved from an early machinery of swimming shared by various eukaryotic cells.
Recruitment of leukocytes from blood vessels to inflamed zones is guided by biochemical and mechanical stimuli, with mechanisms only partially deciphered. Here, we studied the guidance by flow of primary human effector T lymphocytes crawling on substrates coated with ligands of integrins LFA-1 (L2) and VLA-4 (41). We reveal that cells segregate in two populations of opposite orientation for combined adhesion, and show that decisions of orientation rely on a bistable mechanism between LFA-1-mediated upstream and VLA-4-mediated downstream phenotypes. At the molecular level, bistability results from a differential front-rear polarization of both integrins affinity, combined with an inhibiting crosstalk of LFA-1 towards VLA-4. At the cellular level, direction is determined by the passive, flow-mediated orientation of the non-adherent cell parts, the rear uropod for upstream migration and the front lamellipod for downstream migration. This chain of logical events provides a comprehensive mechanism of guiding, from stimuli to cell orientation. STATEMENT OF SIGNIFICANCECell guidance is crucial to many biological functions, but the precise mechanisms remain unclear. We have analyzed here an original phenotype of flow-guided cells mimicking leukocytes crawling onto blood vessels, and show that the controlling parameter of cells decision to migrate upstream or downstream is the relative number of two specific adhesion molecules, the integrins LFA-1 and VLA-4.The spatial polarization of these integrins affinity plus a feedback loop between them creates a bistable system, where cells adhere either by their front or their tail to orient upstream or downstream, respectively. This mechanism proposes a complete chain of events from stimuli to cell orientation which differs strongly from the chemotaxis paradigm, because the external stimuli triggers no signaling.
Permeable agarose barriers allow flow-free gradient generation, applicable to adherent and non-adherent (swimming) cells, as well as co-culture experiments.
We report the design, fabrication and evaluation of an array of microdevices composed of high aspect ratio PDMS pillars, dedicated to the study of tumour spheroid mechanical properties. The principle of the microdevice is to confine a spheroid within a circle of micropillars acting as peripheral flexible force sensors. We present a technological process for fabricating high aspect ratio micropillars (300 μm high) with tunable feature dimensions (diameter and spacing) enabling production of flexible PDMS pillars with a height comparable to spheroid sizes. This represents an upscale of 10 along the vertical direction in comparison to more conventional PDMS pillar force sensors devoted to single cell studies, while maintaining their force sensitivity in the same order of magnitude. We present a method for keeping these very high aspect ratio PDMS pillars stable and straight in liquid solution. We demonstrate that microfabricated devices are biocompatible and adapted to long-term spheroid growth. Finally, we show that the spheroid interaction with the micropillars' surface is dependent on PDMS cellular adhesiveness. Time-lapse recordings of growth-induced micropillars' bending coupled with a software program to automatically detect and analyse micropillar displacements are presented. The use of these microdevices as force microsensors opens new prospects in the fields of tissue mechanics and pharmacological drug screening.
Cell guidance by anchored molecules, or haptotaxis, is crucial in development, immunology and cancer. Adhesive haptotaxis, or guidance by adhesion molecules, is well established for mesenchymal cells such as fibroblasts, whereas its existence remains unreported for amoeboid cells that require less or no adhesion in order to migrate. We show that, in vitro, amoeboid human T lymphocytes develop adhesive haptotaxis mediated by densities of integrin ligands expressed by high endothelial venules. Moreover, lymphocytes orient towards increasing adhesion with VLA-4 integrins (also known as integrin α4β1), like all mesenchymal cells, but towards decreasing adhesion with LFA-1 integrins (also known as integrin αLβ4), which has not previously been observed. This counterintuitive ‘reverse haptotaxis’ cannot be explained by existing mechanisms of mesenchymal haptotaxis involving either competitive anchoring of cell edges under tension or differential integrin-activated growth of lamellipodia, because they both favor orientation towards increasing adhesion. The mechanisms and functions of amoeboid adhesive haptotaxis remain unclear; however, multidirectional integrin-mediated haptotaxis might operate around transmigration ports on endothelia, stromal cells in lymph nodes, and inflamed tissue where integrin ligands are spatially modulated.
Mammalian cells developed two main migration modes. The slow mesenchymatous mode, like fibroblasts crawling, relies on maturation of adhesion complexes and actin fiber traction, while the fast amoeboid mode, observed exclusively for leukocytes and cancer cells, is characterized by weak adhesion, highly dynamic cell shapes, and ubiquitous motility on 2D and in 3D solid matrix. In both cases, interactions with the substrate by adhesion or friction are widely accepted as a prerequisite for mammalian cell motility, which precludes swimming. We show here experimentally and computationally that leukocytes do swim, and that propulsion is not fueled by waves of cell deformation but by a rearward and inhomogeneous treadmilling of the cell envelope. We model the propulsion as a molecular paddling by transmembrane proteins linked to and advected by the actin cortex, whereas freely diffusing transmembrane proteins hinder swimming. This mechanism explains that swimming is five times slower than the cortex retrograde flow. Resultantly the ubiquitous ability of mammalian amoeboid cells to migrate in various environments can be explained for lymphocytes by a single machinery of envelope treadmilling.
Growing multicellular spheroids recapitulate many features of expanding microtumours, and therefore they are an attractive system for biomechanical studies. Here, we report an original approach to measure and characterize the forces exerted by proliferating multicellular spheroids. As force sensors, we used high aspect ratio PDMS pillars arranged as a ring that supports a growing breast tumour cell spheroid. After optical imaging and determination of the force application zones, we combined 3D reconstruction of the shape of each deformed PDMS pillar with the finite element method to extract the forces responsible for the experimental observation. We found that the force exerted by growing spheroids ranges between 100nN and 300nN. Moreover, the exerted force was dependent on the pillar stiffness and increased over time with spheroid growth.
SignificanceCellular guidance is crucial to many biological functions, but the precise mechanisms remain unclear.We have analyzed here an original phenotype of flow-guided cells mimicking leukocytes crawling into the blood vessels and showed that the controlling parameter of cells decision to migrate upstream or downstream was the relative number of two specific adhesion molecules, the integrins LFA-1 and VLA-4. The spatial polarisation of integrins affinity and an intermutually feedback of their activation create a bistable system where cells adhere either by their tip or their tail and orient respectively downstream or upstream. This mechanism therefore proposes a complete chain of event from stimuli to cell orientation and differs strongly from the chemotaxis paradigm because stimuli trigger no signaling. AbstractThe recruitment of leukocytes from blood vessels to inflamed zones is guided by biochemical and mechanical stimuli, with mechanisms only partially deciphered. We studied here the guidance by flow of primary human effector T lymphocytes crawling on substrates coated with ligands of integrins LFA-1 (L2) and VLA-4 (41), and showed that cells segregated in two populations of opposite orientation for combined adhesion. Sharp decisions of orientation were shown to rely on a bistable mechanism between LFA-1-mediated upstream and VLA-4-dominant downstream phenotypes. At the molecular level, bistability results from a differential front-rear polarization of both integrins affinity, combined with an inhibiting crosstalk of LFA-1 toward VLA-4. At the cellular level, directivity with or against the flow is mechanically mediated by the passive orientation of detached uropod or lamellipod by flow. This complete chain of logical events provides a unique mechanistic picture of a guiding mechanism, from stimuli to cell orientation.
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