Global coordination of protrusive forces in migrating immune cells

Background

Cell migration has traditionally been divided into an amoeboid and mesenchymal mode of migration, mechanistically distinct. The amoeboid mode is characterized by a rounded and flexible cell shape, low adhesive interactions and fast motion. While the mesenchymal mode, is based on actin polymerization at the leading edge (lamellipodia), higher adhesive interactions and proteolytic activity. Environmental factors—such as confinement, adhesion levels, and cell-extracellular matrix interactions—play a crucial role in determining migration behavior ^1,2. These factors blur the frontier between the two modes of migration, as cells can transition between them depending on external cues. This plasticity allows cells to adapt and continue migrating despite complex microenvironments³. It is particularly relevant in the context of homeostatic dendritic cell (DC) migration where DCs migrate from peripheral tissues to lymph nodes to present antigens to T Cells and trigger further immune response. DCs employ the amoeboid locomotion, searching for the path of least hindrance to patrol through tight interstitial spaces and confined tissues to finally reach the lymph nodes and inform the immune system.

In the last decade^4–6, studies have shed light on the versatile role of the actin cytoskeleton in driving both pulling and pushing forces during migration. Pulling forces are traditionally associated with mesenchymal-like migration, involving cell-matrix adhesion formation. However, pushing forces—particularly relevant for amoeboid cells—are less explored⁷. Indeed, it remains unclear how these forces are coordinated and contribute to deforming the environment and propelling the cell forward.

The authors aimed to address the current gap in understanding pushing force generation by focusing on the intrinsic mechanism allowing cells to translate intracellular forces into coordinated movement of the entire cell body. Using fast confocal imaging and pushing force microscopy, they offer a fresh perspective on how actin is recycled at the cellular scale to generate movement and synchronize mechanical forces across the cell.

Key findings

• Confinement drives a shift from amoeboid to mesenchymal migration in moving DCs

Reis-Rodrigues and colleagues studied mature DC migration using collagen gels of varying densities and microfluidic channels under chemotactic conditions. In low-density gels and straight channels, the nucleus led the way, while in high-density gels and small constrictions, the centrosome moved first with actin accumulation at the site of constriction, indicating a shift from amoeboid to mesenchymal migration in tighter spaces (Fig. 1A). Cell body compression experiments under stiff and soft gels revealed the formation of a circular actin pool at the center of the cell body (Fig. 1B, C). Under stiffer gels, the actin pool shifted ahead of the nucleus, with key organelles repositioning, indicating internal reorganization in response to mechanical stress (Fig. 1A).

• The central actin pool acts as a lever to clear the path for organelles and the nucleus

The team developed an innovative tool called Pushing force microscopy that enables to assess the precise response of the actin pool to vertical compression. This technique involves agarose mixed with fluorescent beads tracked with sub-micrometer precision using kymographic analysis of rapid confocal microscopy. It allows one to correlate bead displacement with cellular structure and movement. In the context of this study, cross-correlation analysis revealed that the actin pool pushes forward first, followed by the nucleus. Thus, suggesting that the central actin pool serves as a wedge that deforms the environment to limit mechanical stress on the nucleus during migration.

• Cdc42 and its exchange factor DOCK8 are key regulators of the central actin pool

Cdc42 was found to be essential for maintaining the central actin pool. Indeed, inhibiting Cdc42 reduced the pool by 50% without affecting overall F-actin, highlighting its critical role. Additionally, DOCK8, a guanine exchange factor, is also key in regulating this pool, particularly under confinement.

• Central actin communicates with leading edge actin and DOCK8 has a differential impact depending on environmental conditions

Interestingly, DOCK8-/- cells showed no change in migration speed. However, they exhibited smaller deformations, increased nuclear load, and a hyper stabilized leading edge. In confined environments, such as pillar mazes, actin accumulation in the central pool correlated with lamellipodia retraction, while active migration reduced the pool. Thus, actin shifted to the center of the cell under confinement, balancing pushing and protrusive forces while being recycled for coordination. DOCK8-/- cells struggled in confined spaces, but in straight microfluidic channels they migrated faster revealing the central actin pool’s role in efficient migration under mechanical stress.

Bibliography

1. Liu, Y.-J. et al. Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells. Cell 160, 659–672 (2015).
2. Graziani, V., Rodriguez-Hernandez, I., Maiques, O. & Sanz-Moreno, V. The amoeboid state as part of the epithelial-to-mesenchymal transition programme. Trends in Cell Biology 32, 228–242 (2022).
3. Friedl, P. & Wolf, K. Plasticity of cell migration: a multiscale tuning model. J Cell Biol 188, 11–19 (2010).
4. Doyle, A. D., Sykora, D. J., Pacheco, G. G., Kutys, M. L. & Yamada, K. M. 3D mesenchymal cell migration is driven by anterior cellular contraction that generates an extracellular matrix prestrain. Developmental Cell 56, 826-841.e4 (2021).
5. Wang, W. Y., Davidson, C. D., Lin, D. & Baker, B. M. Actomyosin contractility-dependent matrix stretch and recoil induces rapid cell migration. Nat Commun 10, 1186 (2019).
6. Stöberl, S. et al. Nuclear deformation and dynamics of migrating cells in 3D confinement reveal adaptation of pulling and pushing forces. Science Advances 10, eadm9195 (2024).
7. Yamada, K. M. & Sixt, M. Mechanisms of 3D cell migration. Nat Rev Mol Cell Biol 20, 738–752 (2019).

What I like about this preprint

In line with previous work from the authors, this preprint highlights the versatile role of the actin cytoskeleton in generating forces that drive cell migration. It offers a novel perspective on the mechanisms of cell movement by providing a more comprehensive view of how forces are balanced across the entire cell. The study also uncovers an unexpected behavior, the transition from amoeboid to mesenchymal migration, challenging the traditional paradigm. Additionally, the authors employ an innovative experimental approach, pushing force microscopy, which allows them to capture single-cell side views and gain deeper insights into the mechanical forces at play during migration. I was personally inspired to look at mechanisms at the scale of the entire cell raising questions about how actin pools communicate during migration to allow radical cell shape changes through recruitment of actin at the cell cortex for example.

Questions for the authors

1. The detailed molecular mechanisms by which Cdc42 and DOCK8 regulate the formation and dynamics of the central actin pool remain to be uncovered. What would be the pathway to investigate first?  Are there other signaling pathways or regulatory proteins that could be involved?
2. Is there a specific height at which the actin pool is recruited at the center? Is it defined by the nucleus or is it completely independent of the nucleus?
3. Does the altered intracellular organization, due to the central actin pool, have any impact on other cellular processes in DCs, such as antigen processing and presentation or cytokine secretion?
4. The study focuses on DCs. Do other immune cells, such as macrophages or neutrophils, also exhibit this confinement-driven shift in migration mode and the formation of a central actin pool?
5. In your model, the leading edge and the central actin pool communicate, and actin is redistributed between the two locations. Is it possible to imagine an experiment in which you would observe the trafficking of actin monomers between the central pool and the leading edge? I was wondering if it is direct exchange of actin or if it is activation of preexisting actin monomer polymerization. And something else, connects the two locations.

Tags: actin, cell migration, immune cell migration, pushing force microscopy

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