Immune cells were long thought to migrate through tissues in a fast, integrin-independent amoeboid mode that generates little matrix traction. Czerwinski et al. (bioRxiv 2023, published in PNAS 2026) showed that NK92 natural killer cells — with similar burst-like contractile behavior in primary B, T, and NK cells, neutrophils, and monocytes — intermittently switch to a contractile, mesenchymal-like migration mode when facing pores narrower than the cell diameter in 3D collagen networks and human amniotic membrane tissue: brief actomyosin-driven, integrin β1/β2-mediated traction bursts of up to ~100 nN keep cells from getting stuck, and cells that cannot contract or adhere lose motility. What remains unresolved is the mechanism and regulation of this switch: which adaptor and signaling molecules couple actomyosin contraction to the integrins, whether switching is triggered by active sensing of local steric hindrance (pore size, stiffness) or occurs stochastically, how traction forces and nuclear deformability jointly enable passage through narrow pores, how the machinery is regulated on long time scales (e.g., the transient benefit of microtubule destabilization reverting after ~18 hours). Whether the switch also operates in vivo, notably during immune-cell infiltration of dense tumor stroma, is raised by the source as a contingent direction rather than as part of this mechanistic problem.

Background

To reach targets outside the bloodstream, leukocytes must migrate through connective tissue, which consists largely of a three-dimensional (3D) network of extracellular matrix (ECM) biopolymers such as collagen, whose pore sizes can be smaller than a cell's diameter — a physical obstacle termed steric hindrance. Two canonical single-cell migration modes are distinguished. Amoeboid migration is fast (up to about 10 µm/min), relies on shape change and low-friction, non-specific interactions with the matrix, and classically does not require integrins (the transmembrane receptor family that binds ECM proteins such as collagen and fibronectin) or large traction forces (the pulling forces a cell exerts on its surroundings). Mesenchymal migration is slower, integrin-adhesion-dependent, and generates substantial traction forces to deform the surrounding fibers. Lämmermann et al. (Nature, 2008) established rapid integrin-independent 'flowing and squeezing' as the default interstitial leukocyte mode, and Wolf et al. (J Cell Biol, 2003) had earlier shown that cancer cells can compensate for lost proteolytic matrix degradation by switching between mesenchymal and amoeboid modes, establishing migration-mode plasticity. Renkawitz et al. (Nat Cell Biol, 2009) showed that even amoeboid dendritic cells transmit adaptive forces, and Yamada and Sixt (Nat Rev Mol Cell Biol, 2019) framed 3D migration as a continuum in which adhesiveness and confinement jointly determine the mode. Against this background, Czerwinski et al. measured simultaneous migration dynamics and matrix-deforming contractility (via time-lapse confocal reflection microscopy and 3D traction force microscopy, i.e. reconstruction of cell-generated forces from measured matrix deformations) for NK92 natural killer cells (an immortalized NK-cell line), ex-vivo expanded primary NK cells, and primary blood immune cells in collagen gels of controlled stiffness and pore size, and in fibrous human amniotic membrane tissue. They found intermittent contractile bursts (up to ~100 nN, roughly comparable to mesenchymal cells after accounting for cell size) whose timing coincides with passage through narrow pores; actomyosin inhibition (blebbistatin, ROCK inhibitor) or adhesion interference (pluronic-coated fibers, non-adhesive carbomer gels, EDTA) leaves the speed of still-motile cells unchanged but drastically reduces the fraction of motile cells; blocking integrins β1 or β2 reduces motile fraction and directional persistence (β2 more strongly, matching its higher expression); and an integrin β1 activating antibody does not improve motility, indicating tight regulation. Nocodazole-induced microtubule depolymerization transiently raises forces and motile fraction, reverts after ~18 hours, and confounds two effects — increased nuclear deformability and increased contractility — that the paper cannot separate. Caillier et al. (J Cell Biol, 2024) subsequently showed that activated Th1 T cells under confinement assemble canonical focal adhesion proteins (integrins, talin, vinculin) at sites of traction stress, indicating that the molecular force-transmission machinery of confined T cells is mesenchymal-like — but for NK cells in 3D biopolymer networks the coupling molecules remain unidentified, the steric-hindrance trigger is supported only by correlational evidence, and the in-vivo relevance of the mode is unverified.

Problem Statement

The problem that remains open after Czerwinski et al. (bioRxiv 2023; PNAS 2026) is the mechanism and regulation of the steric-hindrance-induced switch from amoeboid to integrin-mediated contractile migration in immune cells moving through 3D biopolymer matrices. Its jointly linked facets are: (i) identify the molecular machinery — adaptor and signaling proteins downstream of integrins β1 and β2 — by which actomyosin contractile force is transmitted to the extracellular matrix during the contractile bursts (the source data implicate the integrins but identify none of the couplers; the paper notes that the mechanism of force transmission across the plasma membrane "has not yet been characterized in these cells"); (ii) causally establish the trigger of the switch: the source's data support its hypothesis that the switch from an amoeboid to a contractile migration mode is triggered when the cell encounters increased steric hindrance, but that evidence is correlational, and it remains to be determined whether switching reflects active sensing of local steric hindrance such as pore size or matrix stiffness, or occurs stochastically — for example by live-cell force imaging combined with simultaneous mapping of the matrix topology immediately around each cell; (iii) disentangle the relative contributions of nuclear deformability and traction forces to passage through narrow pores, since perturbations such as nocodazole increase both simultaneously; and (iv) determine how the adhesion/contractility machinery is regulated on long time scales, including why the short-term benefit of microtubule destabilization reverts into loss of motility after ~18 hours. These facets share one objective — a causal mechanistic account of how and when immune cells switch migration modes to overcome steric hindrance, and how this machinery is regulated. Two further directions are noted by the source but framed there as contingent rather than as parts of the open mechanistic question: whether integrin activity can be tuned to improve immune-cell infiltration into dense microenvironments such as tumor stroma (raised in the paper as a translational implication), and direct in-vivo verification that the contractile, integrin-dependent mode operates in living tissue, which would require intravital imaging stable enough to detect matrix deformations in the range of tens of nanometres — described by the authors as currently unfeasible, though future improvements may enable a direct verification.

References

  1. Czerwinski T, Bischof L, Böhringer D, Kara S, Wittmann E, Winterl A, Gerum R, Nusser G, Wiesinger M, Budday S, Lux A, Voskens C, Fabry B, Mark C. Immune cells employ traction forces to overcome steric hindrance in 3D biopolymer networks. bioRxiv preprint, 2023. doi:10.1101/2023.04.20.537658. https://www.biorxiv.org/content/10.1101/2023.04.20.537658v1
  2. Czerwinski T, Bischof L, Böhringer D, Kara S, Strissel PL, Strick R, Huhn N, Winterl A, Gerum R, Wittmann E, Schneider M, Beckmann MW, Nusser G, Wiesinger M, Budday S, Lux A, Voskens C, Fabry B, Mark C. Immune cells employ intermittent integrin-mediated traction forces for 3D migration. Proceedings of the National Academy of Sciences USA, 2026, 123(11):e2524427123. doi:10.1073/pnas.2524427123. https://doi.org/10.1073/pnas.2524427123 (published version of the bioRxiv preprint above)
  3. Caillier A, Oleksyn D, Fowell DJ, Miller J, Oakes PW. T cells use focal adhesions to pull themselves through confined environments. The Journal of Cell Biology, 2024, 223(10). doi:10.1083/jcb.202310067. https://doi.org/10.1083/jcb.202310067
  4. Böhringer D, Cóndor M, Bischof L, Czerwinski T, Gampl N, Ngo PA, Bauer A, Voskens C, López-Posadas R, Franze K, Budday S, Mark C, Fabry B, Gerum R. Dynamic traction force measurements of migrating immune cells in 3D biopolymer matrices. Nature Physics, 2024, 20:1816-1823. doi:10.1038/s41567-024-02632-8. https://doi.org/10.1038/s41567-024-02632-8
  5. Reis-Rodrigues P, Canigova N, Avellaneda MJ, Gaertner F, Vaahtomeri K, Riedl M, Merrin J, Hauschild R, Fukui Y, Juanes Garcia A, Sixt M. Global coordination of protrusive forces in migrating immune cells. bioRxiv preprint, 2024. doi:10.1101/2024.07.26.605242. https://www.biorxiv.org/content/10.1101/2024.07.26.605242v1
  6. Ucla P, Lê-Chesnais J, Ver Hulst H, Ju X, Calvente I, Nematollahi E, Leconte L, Salamero J, Bonnet I, Monnot C, Moreau HD, Landoussi J, Semetey V, Coscoy S. Quantifying cell traction forces at the single-fiber scale in 3D: An approach based on deformable photopolymerized fiber arrays. Proceedings of the National Academy of Sciences USA, 2025, 122(42). doi:10.1073/pnas.2507677122. https://doi.org/10.1073/pnas.2507677122
  7. Schwartz AB, Kandasamy A, Tu Y, de Lecea A, Nigam V, del Álamo JC, Yeh YT. Neutrophils exhibit distinct migration phenotypes that are modulated by transendothelial migration. Communications Biology, 2026, 9. doi:10.1038/s42003-026-10122-1. https://doi.org/10.1038/s42003-026-10122-1
  8. Villella C, Ciccioli M, Anton IM, Calle Y. Plasticity in leukocyte migration during haematopoiesis and inflammation. Journal of Muscle Research and Cell Motility, 2025, 46:135-151. doi:10.1007/s10974-025-09691-1. https://doi.org/10.1007/s10974-025-09691-1
  9. Lämmermann T, Bader BL, Monkley SJ, Worbs T, Wedlich-Söldner R, Hirsch K, Keller M, Förster R, Critchley DR, Fässler R, Sixt M. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature, 2008, 453:51-55. doi:10.1038/nature06887. https://doi.org/10.1038/nature06887
  10. Wolf K, Mazo I, Leung H, Engelke K, von Andrian UH, Deryugina EI, Strongin AY, Bröcker EB, Friedl P. Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. The Journal of Cell Biology, 2003, 160:267-277. doi:10.1083/jcb.200209006. https://doi.org/10.1083/jcb.200209006
  11. Renkawitz J, Schumann K, Weber M, Lämmermann T, Pflicke H, Piel M, Polleux J, Spatz JP, Sixt M. Adaptive force transmission in amoeboid cell migration. Nature Cell Biology, 2009, 11:1438-1443. doi:10.1038/ncb1992. https://doi.org/10.1038/ncb1992
  12. Yamada KM, Sixt M. Mechanisms of 3D cell migration. Nature Reviews Molecular Cell Biology, 2019, 20:738-752. doi:10.1038/s41580-019-0172-9. https://doi.org/10.1038/s41580-019-0172-9