When leukocytes migrate through dense tissues whose pores are too small for free passage, they reposition the nucleus behind the centrosome and organelles and assemble a mechanosensitive central pool of polymerized actin between the cell front and the nucleus. This pool, controlled by the Cdc42 guanine-nucleotide-exchange factor DOCK8 together with WASp and other Cdc42 effectors, pushes outward to dilate a path for organelles and the nucleus, and is kept in a homeostatic balance with leading-edge protrusion. A companion study has already established that the pool protects the nucleus (limiting nuclear deformation, DNA damage, and senescence) and that the Hippo kinase MST1 is a necessary pathway component in T cells. What remains open is the mechanism: the identity of the mechanosensitive pathway upstream of DOCK8 (what senses cell compression and how MST1 and DOCK8 are engaged) and the mode of communication between the central and leading-edge actin pools (for example actin-monomer competition, signaling feedback, or mechanical coupling).

Background

Immune cells patrol tissues by amoeboid migration: a fast, adhesion-independent mode of locomotion in which the cell squeezes through pre-existing matrix pores. The nucleus is the largest and stiffest organelle and constitutes the physical bottleneck of passage through narrow pores (Wolf et al. 2013); in loosely porous environments, motile leukocytes such as dendritic cells (DCs) use the forward-positioned nucleus as a mechanical probe that follows the path of least resistance (Renkawitz et al. 2019). When even the largest pores preclude free passage, leukocytes switch polarity: the nucleus is swept to the back while the centrosome (the microtubule-organizing centre, MTOC) and the bulk of organelles move in front. Reis-Rodrigues, Avellaneda, Canigova and co-workers (Nature Immunology 2025; bioRxiv preprint 2024) showed that this reconfiguration is accompanied by local, compression-triggered assembly of a central F-actin pool located between the leading edge and the nucleus, which pushes outward to transiently dilate a path for the organelles and the nucleus. DOCK8 is an atypical guanine-nucleotide-exchange factor (GEF, an enzyme that activates small GTPases by promoting GDP-to-GTP exchange) for the Rho-family GTPase Cdc42; it is expressed mainly in hematopoietic cells, and loss-of-function mutations in humans cause a combined immunodeficiency in which leukocytes become entangled and fragment when migrating through dense tissues (Zhang et al. 2014; Harada et al. 2012 showed DOCK8 is the Cdc42 activator critical for interstitial DC migration). DOCK8 colocalizes with the central actin pool, and its loss abolishes the pool while actin accumulates instead at the leading edge, producing an 'unleashed' front that detaches from the trapped cell body. Cdc42 activates WASp (Wiskott–Aldrich syndrome protein), a nucleation-promoting factor for the Arp2/3 complex, which builds branched actin networks; Gaertner et al. (2022) had earlier described WASp-triggered mechanosensitive actin patches that help leukocytes cross dense tissues, and in the 2025 paper WASp is recruited to the central pool and contributes to, but is not essential for, its formation. The central and front actin pools are anti-correlated: experimentally depleting the central pool enhances protrusion at the front, speeding cells up in permissive environments but impairing them in restrictive ones, so the two pools form a regulatory loop that balances path dilation against advancement of the leading edge. The Hippo pathway kinase MST1 (mammalian Sterile 20-like kinase 1, also called STK4), better known for controlling organ size through cell proliferation, is an upstream activator of DOCK8 and, as the companion study below shows, a necessary component of this mechanosensitive pathway in T cells; whether MST1 directly activates or positions DOCK8 in this context is not established. A companion study (Shen et al., Science Immunology 2025) demonstrated a DOCK8-dependent, mechanoresponsive central actin pool in mouse and human T cells, identified MST1 as a necessary component of the pathway, and showed that loss of the pool increases nuclear deformation, DNA damage, and premature senescence — confirming the nuclear-protection hypothesis. In yeast, the two F-actin structures (patches and cables) communicate by competing for a shared pool of actin monomers, but how distinct actin pools communicate in animal cells is poorly understood, and the upstream mechanosensing and pool-coupling mechanisms remain unresolved.

Problem Statement

Elucidate the molecular mechanism by which confined migrating leukocytes assemble the DOCK8-dependent central actin pool and coordinate it with leading-edge protrusion. Concretely: (1) identify the mechanosensitive pathway upstream of DOCK8 — the pathway that Reis-Rodrigues et al. (2025) state 'remains to be identified' — that is, which molecular entity senses compression of the cell body (at the plasma membrane, the actomyosin cortex, the nuclear envelope, or another site), in what order MST1, DOCK8, Cdc42, and the downstream actin nucleators act, and whether MST1 directly activates or positions DOCK8 in this context; and (2) determine how the central actin pool and the leading-edge actin pool communicate — a question Reis-Rodrigues et al. (2025) state is 'poorly understood in animal cells' — for example through competition for a shared pool of monomeric (G-)actin as in yeast, through signaling feedback, through mechanical coupling, through a combination, or through another mechanism. A complete answer must address every part above: the compression-sensing entity and its site; the order of action of MST1, DOCK8, Cdc42, and the downstream actin nucleators; whether MST1 directly activates or positions DOCK8; and the mode by which the central and leading-edge actin pools communicate. Addressing only the upstream pathway, or only the pool coupling, is a partial answer that does not resolve the task. Already established and not part of what needs to be shown: the existence and compression-triggered assembly of the central pool and its DOCK8 dependence, the anti-correlation between the central and leading-edge pools, the DOCK8-dependent mechanoresponsive pool in mouse and human activated T cells with MST1 as a necessary pathway component, the pool's nuclear-protection function (limiting nuclear deformation, DNA damage, and senescence), and the results-level finding that WASp-triggered actin polymerization contributes to, but is not essential for, central-pool formation.

Related questions that are not part of the required mechanistic account include how the central pool cooperates with the WASp-mediated mechanosensitive actin patches described by Gaertner et al. (2022), and whether and how the pool operates in vivo and across leukocyte subsets (such as neutrophils, dendritic-cell subsets, and T-cell subsets) in which its dependence on DOCK8 and MST1 may differ.

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