Mechanosensitivity of amoeboid cells crawling in 3D

Florian Gaertner, Patricia Reis-Rodrigues, Ingrid de Vries, Miroslav Hons, Juan Aguilera, Michael Riedl, Alexander Leithner, Jack Merrin, Vanessa Zheden, Walter Anton Kaufmann, Robert Hauschild, Michael Sixt

Preprint posted on May 12, 2021

A push in the orthogonal direction: Mechanosensing by amoeboid cells

Selected by Jessica L. Teo


Cell migration plays an essential role in a multitude of physiological processes such as embryogenesis, tissue regeneration, immune surveillance, and cancer metastasis (Friedl and Gilmour, 2009; Friedl and Wolf, 2003; Krummel et al., 2016; Scarpa and Mayor, 2016; Worbs et al., 2017). In vivo, cells are confronted with a complex and confining 3-dimensional (3D) microenvironment comprising other cell types and the extracellular matrix. To migrate efficiently, cells need to navigate their way through this microenvironment while maintaining nuclear integrity and viability (Davidson et al., 2014; Petrie et al., 2014; Wolf et al., 2013).

Although extracellular matrix composition, topography, and elasticity have been observed to significantly influence cell migration (Pickup et al., 2014; Wolf and Friedl, 2011; Yamada et al., 2019), cells also actively sense and remodel the surrounding matrix to facilitate motility (Doyle et al., 2015; Provenzano et al., 2008). For instance, during 3D mesenchymal cell migration, cells employ a combination of lamellipodial protrusions and integrin-based focal adhesions to probe the geometry and mechanical properties of the matrix (Plotnikov and Waterman, 2013). As they migrate, they remodel the matrix by degrading matrix proteins with proteases and by physically deforming matrix components (Doyle et al., 2021; Yamada and Sixt, 2019). However, the mechanism behind the migration of amoeboid cells, such as immune cells, in confined 3D microenvironments remains unclear. Amoeboid cells migrate rapidly by adapting to their environment rather than remodeling it and they do so with minimal adhesions (Hons et al., 2018; Liu et al., 2015; Wolf et al., 2003). Recent studies indicate that by actively changing their shape, amoeboid cells probe their environment and identify larger pores over smaller ones (Renkawitz et al., 2019). If the cell must migrate through a smaller pore, it actively deforms itself while transiently dilating the pore. Despite these observations, how amoeboid cells sense the forces imposed on them, and how they counteract these forces for motility are still underinvestigated.

In this new preprint, Gaertner et al. investigate how immune cells mechanosense in 3D and respond to forces by applying extrinsic mechanical load, challenging cells to restrictive environments as well as correlating their observations with an in vivo lymph node homing assay.


Key findings

To understand how amoeboid cells respond to compressive loads of varying stiffness, the authors overlayed dendritic cells isolated from mice with non-degradable agarose casts within the range of physiological stiffness. Under this mechanical load, with increasing stiffness (low: 2.5kPa; intermediate: 10kPa, high: 17.5kPa), motility decreased, and cells started to transit from the typical continuous mode of migration to a stop-and-go pattern of movement. Remarkably, dendritic cells migrating under agarose of intermediate stiffness generated small actin-rich foci dispersed across the cell area with substantial accumulation around the cell body. To determine if cell adhesion with the underlying substrate is required for the formation of these actin foci, the authors cultured cells between passivated coverslips and the agarose cast. Under this condition, actin foci continued to develop demonstrating that cell-substrate adhesions are not required for generating these distinct actin hubs. Over time, these actin foci formed elongated stripes. Using confocal microscopy and correlated light and scanning electron microscopy, the authors observed that the stripes were spike-like surface structures that were extending into the agarose cast (Figure 1).

Figure 1. Scanning electron microscopy images of a dendritic cell overlayed with a 10kPa agarose gel. Upper panel shows a section across the leading edge of the cell. Lower panel shows a section across the cell body containing the nucleus. Blue dotted line outlines the nucleus and black arrow heads denote actin spikes formed vertically. Adapted from Figure 1J (Gaertner et al. 2021).

To determine the molecular players involved in the formation of these actin foci/spikes, the authors inhibited formins and Arp2/3- actin nucleators that regulate the cortical actin cytoskeleton. While formin inhibition with SMIFH2 showed a slight reduction in cell motility, Arp2/3 inhibition with CK666 resulted in a substantial decrease in motility with cells spending most of the time in arrest. Even though the Arp2/3 complex can be activated by nucleation promoting factors of the Wiskott-Aldrich Syndrome protein (WASp) and WASp-family verprolin homologue protein (WAVE) families, depletion of WASp and not the WAVE complex perturbed actin foci and spikes formation in dendritic cells migrating under agarose of intermediate stress. In addition, WASp-EGFP in dendritic cells formed punctate structures closely mimicking the distribution and flow pattern of actin foci. Importantly, cross-correlation of the number of actin foci with speed revealed that foci formation preceded cell acceleration by 1-2 min. This suggests that actin foci could act as prerequisite cellular structures assembled to lift the load imposed by the agarose to facilitate cell migration.

Next, the authors proceeded to test their observations in physiological environments using an in vivo lymph node homing assay. To do so, they injected both Wild-Type (WT) and WASp-/- dendritic cells into the footpads of WT recipient mice. 24h later, they measured the recruitment of these dendritic cells to draining popliteal lymph nodes. Strikingly, recruitment of WASp-/- dendritic cells was significantly reduced compared to WT controls. Next, to test if a similar mechanism is at play in other immune cell types, the authors purified WASp-/- naïve T cells from mice. Consistent with their observations in confined dendritic cells, WT T cells assembled highly dynamic actin foci when overlayed with agarose casts of intermediate stiffness. These foci were almost depleted in WASp-/- T cells. Next, the authors investigated the ability of WASp-/- T cells to home to popliteal lymph nodes. Interestingly, even though WASp-/- T cells were able to migrate successfully to the lymph nodes, they exhibited reduced mean speed. Together, these data show that cells of both myeloid and lymphoid hematopoietic lineages adopt vertical WASp-driven protrusions for 3D migration under compressive load. Finally, WASp-deficient patients display congenital immunodeficiency resulting from defective T cell functions. These observations provide mechanistic insights into the impaired cell migration noted in X-linked WAS.

What I like about this preprint

In this study, the authors tackled the question of how amoeboid cells mechanosense and respond to extrinsic forces in 3D. Using nifty reductionist approaches and an in vivo lymph node assay, they discovered that cells manage to counteract extrinsic forces by activating WASp-mediated actin polymerization. This results in the assembly of actin spikes enabling the  cells to ‘lift’ the load before migrating. I like how the authors used clever approaches to dissect the mechanism behind 3D migration in response to forces.

Questions for the authors

  1. The observation that cells assemble vertical actin spikes to push the load away is interesting. I’m curious if these spikes occur at a higher frequency/density around the nucleus?
  2. I wonder if WASp-/- dendritic cells would show similar defects in homing to draining popliteal lymph nodes when they are injected together with WT dendritic cells into the footpad of WASP-/- mice?


Davidson, P.M., Denais, C., Bakshi, M.C., and Lammerding, J. (2014). Nuclear deformability constitutes a rate-limiting step during cell migration in 3-D environments. Cell Mol Bioeng 7, 293-306.

Doyle, A.D., Carvajal, N., Jin, A., Matsumoto, K., and Yamada, K.M. (2015). Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions. Nat Commun 6, 8720.

Doyle, A.D., Sykora, D.J., Pacheco, G.G., Kutys, M.L., and Yamada, K.M. (2021). 3D mesenchymal cell migration is driven by anterior cellular contraction that generates an extracellular matrix prestrain. Dev Cell 56, 826-841 e824.

Friedl, P., and Gilmour, D. (2009). Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Biol 10, 445-457.

Friedl, P., and Wolf, K. (2003). Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 3, 362-374.

Hons, M., Kopf, A., Hauschild, R., Leithner, A., Gaertner, F., Abe, J., Renkawitz, J., Stein, J.V., and Sixt, M. (2018). Chemokines and integrins independently tune actin flow and substrate friction during intranodal migration of T cells. Nat Immunol 19, 606-616.

Krummel, M.F., Bartumeus, F., and Gerard, A. (2016). T cell migration, search strategies and mechanisms. Nat Rev Immunol 16, 193-201.

Liu, Y.J., Le Berre, M., Lautenschlaeger, F., Maiuri, P., Callan-Jones, A., Heuze, M., Takaki, T., Voituriez, R., and Piel, M. (2015). Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells. Cell 160, 659-672.

Petrie, R.J., Koo, H., and Yamada, K.M. (2014). Generation of compartmentalized pressure by a nuclear piston governs cell motility in a 3D matrix. Science 345, 1062-1065.

Pickup, M.W., Mouw, J.K., and Weaver, V.M. (2014). The extracellular matrix modulates the hallmarks of cancer. EMBO Rep 15, 1243-1253.

Plotnikov, S.V., and Waterman, C.M. (2013). Guiding cell migration by tugging. Curr Opin Cell Biol 25, 619-626.

Provenzano, P.P., Inman, D.R., Eliceiri, K.W., Trier, S.M., and Keely, P.J. (2008). Contact guidance mediated three-dimensional cell migration is regulated by Rho/ROCK-dependent matrix reorganization. Biophys J 95, 5374-5384.

Renkawitz, J., Kopf, A., Stopp, J., de Vries, I., Driscoll, M.K., Merrin, J., Hauschild, R., Welf, E.S., Danuser, G., Fiolka, R., et al. (2019). Nuclear positioning facilitates amoeboid migration along the path of least resistance. Nature 568, 546-550.

Scarpa, E., and Mayor, R. (2016). Collective cell migration in development. J Cell Biol 212, 143-155.

Wolf, K., and Friedl, P. (2011). Extracellular matrix determinants of proteolytic and non-proteolytic cell migration. Trends Cell Biol 21, 736-744.

Wolf, K., Muller, R., Borgmann, S., Brocker, E.B., and Friedl, P. (2003). Amoeboid shape change and contact guidance: T-lymphocyte crawling through fibrillar collagen is independent of matrix remodeling by MMPs and other proteases. Blood 102, 3262-3269.

Wolf, K., Te Lindert, M., Krause, M., Alexander, S., Te Riet, J., Willis, A.L., Hoffman, R.M., Figdor, C.G., Weiss, S.J., and Friedl, P. (2013). Physical limits of cell migration: control by ECM space and nuclear deformation and tuning by proteolysis and traction force. J Cell Biol 201, 1069-1084.

Worbs, T., Hammerschmidt, S.I., and Forster, R. (2017). Dendritic cell migration in health and disease. Nat Rev Immunol 17, 30-48.

Yamada, K.M., Collins, J.W., Cruz Walma, D.A., Doyle, A.D., Morales, S.G., Lu, J., Matsumoto, K., Nazari, S.S., Sekiguchi, R., Shinsato, Y., et al. (2019). Extracellular matrix dynamics in cell migration, invasion and tissue morphogenesis. Int J Exp Pathol 100, 144-152.

Yamada, K.M., and Sixt, M. (2019). Mechanisms of 3D cell migration. Nat Rev Mol Cell Biol 20, 738-752.

Tags: actin cytoskeleton, amoeboid motility, cell migration, confinement, leukocyte, mechanical load, wiskott-aldrich syndrome protein

Posted on: 24th June 2021


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