Coordinated in confined migration: The crosstalk between the nucleus and ion channel-mediated mechanosensation

Abstract

Cell surface and intracellular mechanosensors enable cells to perceive different geometric, topographical and physical cues. Mechanosensitive ion channels localized at the cell surface - and on the nuclear envelope- are among the first to sense and transduce these signals. Beyond compartmentalizing the cell’s genome and its transcription, the nucleus also serves as a mechanical gauge of different physical and topographical features of the tissue microenvironment. This review delves into the intricate mechanisms by which the nucleus and different ion channels regulate cell migration in confinement. We review evidence suggesting an interplay between macromolecular nuclear-cytoplasmic transport and ionic transport across the cell membrane during confined migration. We also discuss the roles of the nucleus and ion channel-mediated mechanosensation, whether acting independently or in tandem, in orchestrating migratory mechanoresponses. The understanding of nuclear and ion channel sensing – and their crosstalk- is critical to advancing our knowledge of cell migration in health and disease.

MECHANOSENSORS IN CONFINED MIGRATION

Cell migration regulates diverse physiological and pathological processes, such as embryonic development, wound healing, immune response, and cancer metastasis [1]. Although cell locomotion has historically been studied on 2-dimensional (2D) planar surfaces, it is now well accepted that the 2D environment does not recapitulate the complex geometric and topographical cues that migrating cells encounter in vivo [2]. Microscopy studies have shown that cells in vivo migrate through diverse confining (i.e., spatially restricting) microenvironments, including pores with diameters ranging from ~1 to 20 μm [3], fiber- and channel-like tracks with widths varying from ~3 to 30 μm [4], microvessels smaller than the size of cells [5] and openings between endothelial cells spanning from ~0.5 to 5 μm [6]. Numerous recent studies have aimed to elucidate the modes and mechanisms of confined cell migration [1,2,7–13].

The physical forces applied to cells due to the spatial restrictions of confinement alter signaling pathways, and consequently, their migration modes in a 3D environment [8,14]. During 3D cell migration, physical cues, such as extracellular fluid viscosity [15], hydraulic resistance [16], shear flow [17], the stiffness [18], viscoelasticity [19] and matrix mechanical plasticity [20] of the local microenvironment, compressive forces due to confinement or channel geometry [9,21], all trigger intracellular responses that allow cells to adapt to the diverse landscapes they encounter. Cells process these extracellular stimuli and translate them into biochemical and/or biophysical signals through a process known as mechanosensing or mechanotransduction [2,13,22].

As the largest and stiffest cell organelle, the nucleus poses a rate-limiting barrier to confined cell migration [23,24]. In the absence of extracellular matrix (ECM) proteolysis, migration is halted at pore sizes less than about 10% of the nuclear cross-sectional area (e.g., ~7 μm² in tumor cells) due to cells’ inability to translocate their nuclei through such narrow pores [24]. When the pore size is larger than this limit, the nucleus still remains a barrier to motility and must deform to facilitate migration through confined spaces [25]. However, the nucleus is not passive in this event; it processes mechanical signals and initiates signaling responses that have downstream effects on cell migration [13,22,26]. Besides the nucleus, mechanosensitive ion channels (MICs) may also serve as direct transducers of physical cues [27], while other ion transporters, ion and water channels can mediate mechanosensation indirectly [15]. These channels and molecules are primarily localized at the cell surface, facilitating regulated fluxes of water and ions, including calcium, magnesium, protons, sodium, potassium, and chloride. Such ions can act as secondary messengers (calcium, magnesium) or as regulators of the intracellular ionic concentration, thereby influencing the efficiency, direction, and decision-making strategies of migrating cells [15–17,28–30]. Other key mediators of physical signals include integrin-based focal adhesions, which sense both intracellular and extracellular tension [31–35] as well as ECM stiffness and viscoelasticity [36,37]. For more comprehensive information on the role of cell-matrix interactions in 3D cell migration, we refer the reader to relevant outstanding reviews [2,19,38].

While focusing on migrating cells, we review how the nucleus, MICs, ion transporters, and ion and water channels mediate cell responses to the various geometric and topographical cues of confinement. We first examine how the structural components of the nucleus contribute to mechanosensing pathways, gene expression, mechanoadaptation, and cell migration. Then, we cover the individual and concerted mechanotransductive functions of MICs, ion transporters, and ion channels. Finally, we discuss how they cooperate with the nucleus to facilitate cell migration in confined microenvironments.

THE NUCLEUS IS BOTH A PHYSICAL BARRIER AND A KEY MECHANOSENSOR

The role and mechanisms of nuclear organization in cellular mechanosensation

The nucleus serves as a cellular compartment sequestering genomic DNA, enabling elaborate gene expression controls, and physically protecting the genome. Its exquisite mechanical properties (large size, stiffness) also define the mode and efficiency of cells migrating in confining environments. The nuclear interior, which contains chromatin, many species of RNAs, and proteins in soluble form or organized into nuclear matrix or nuclear bodies [39,40], is physically separated from the rest of the cell by the nuclear envelope (NE) [41]. The NE comprises inner and outer nuclear membranes occupied by numerous membrane-bound or transmembrane proteins (Fig. 1A–B). The passive, diffusion-limited or active, receptor-mediated nuclear-cytoplasmic transport (NCT) of macromolecules occurs through the channels of nuclear pore complexes (NPCs) embedded through both NE layers [41,42]. NPCs are large macromolecular assemblies of more than 500 protein subunits with a molecular mass of 110 MDa (~120 nm in diameter) that are abundantly expressed in most cells (e.g., ~10 per μm² in HeLa cells [43]) [44]. External forces acting on a cell can deform the NE and dilate NPCs (Fig. 1C), thereby enabling passive and facilitated diffusional exchange of nuclear and cytoplasmic constituents [45], which can affect the transcriptional activity and behavior of migrating cells.

Figure 1. Mechanosensation by the nucleus: mechanisms and consequences.

(A) LINC complexes transmit extracellular and cytoplasmic, actin-mediated mechanical forces to the nucleus. (B) Nuclear lamina mechanically supports the nucleus and NE, and its stiffness defines nuclear deformability and resistance in confined migration. LADs or chromatin mediate force. (C) Mechanical force-induced NPC stretching attenuates the passive exclusion limit of NPC channels, enabling passive macromolecular nucleocytoplasmic exchange, including YAP nuclear entry and signaling. (D) Histone modification mediates the interconversion of stiffer heterochromatin and softer euchromatin, contributing to changes in nuclear resistance during confined migration and enabling long-lasting mechanomemory. (E) LLPS refers to the formation of nuclear condensates due to homotypic interactions, which are dispersed by the application of mechanical forces. (F) Posterior actomyosin contractility induced by confinement along the dorsoventral axis leads to elevated pressure at the cell’s rear, passive anterograde fluid flow into the nucleus through NPC channels, nuclear volume expansion, and NE blebbing. (G) Confinement-induced nuclear deformation leads to NE rupture, inducing unregulated mixing of nuclear and cytoplasmic factors. Nuclear entry of ER-associated TREX1 exonuclease causes DNA damage, also exacerbated by the cytoplasmic exit of nuclear DNA repair factors. At the same time, cytoplasmic access to dsDNA can trigger cGAS/STING-dependent signaling.

Crucial to the protection of genomic DNA integrity and at the same time enabling viscoelastic deformation under mechanical stress, the NE is supported by the layer of nuclear lamina [46], which is a tightly woven meshwork comprised of A- (A, C, C2) and B- (B1–3) type lamins (members of the type V intermediate filament proteins) and lamin-binding proteins [25,41,47] (Fig. 1B). Lamins are involved in the regulation of transcription, embryonic development, cell differentiation, DNA replication, cell polarization, migration, and metastasis, among others [23,41,46,48–50]. Many, if not most, of such “regulatory” functions of lamins result from the role of lamina as a structure protecting the genome and profoundly affecting its transcriptional activity. Nuclear lamina organizes chromosomal territories through lamin-associated chromosomal domains (LADs) [51,52], controls NPC positioning [53], mediates cytoplasmic-to-nuclear mechanotransduction via the linker of the nucleoskeleton and cytoskeleton (LINC) complex, and connects NE to the nuclear interior through direct interactions with inner NE proteins (Fig. 1A), including emerin and BAF [51].

Individual lamins may contribute to the mechanical properties of the lamina and nuclear mechanosensation in an isoform-specific manner. Studies in various systems indicated that lamin-A dominantly contributes to nuclear viscosity (resistance to flow) [54] and stiffness (resistance to deformation) [54–56]. Consistent with this view, overexpression of lamin-A increases nuclear stiffness, thereby impairing cell entry and migration in confinement [57,58]. Furthermore, expression of lamin-A progeria mutant, which alters lamina structure due to its farnesylation-dependent association with nuclear envelope [59], increases nuclear stiffness and suppresses cell migration in 3D [60]. On the other hand, depletion of lamin-A/C, which promotes nuclear deformability, facilitates 3D migration through confining pores but reduces cell survival by exposing the nucleus to migration-induced stress due to increased NE rupture and subsequent DNA damage [23,48,61,62].

In contrast to lamin-A, earlier studies suggested that B-type lamins had little or no effect on nuclear stiffness [55]; instead, they primarily conferred nuclear elasticity (spring-like behavior) [54]. Advances in cellular models and measurement techniques have recently refined this view. A study that applied micropipette aspiration and nanoindentation measurements in triple lamin-knockout mouse embryonic fibroblasts (MEFs) reconstituted with individual lamins reached a more nuanced model of the interplay of lamins and chromatin in nuclear mechanical properties [46]. According to this model, while nuclear viscosity is dominantly defined by lamin-A, both A- and B-type lamins contribute to nuclear stiffness and elasticity. At the same time, simultaneous expression of lamin-A and lamin-B1 stabilizes chromatin condensation [46]. Another study utilizing mouse MEFs individually depleted of lamins and an array of measurements, including atomic force microscopy (AFM), confirmed that lamin- B1 and B2, in addition to lamin A, contribute to nuclear stiffness and regulate constricted cell migration [35]. Notably, the levels of lamin-B1 vary across different types of cancers [63], and its increased expression predicts poor prognosis in many cancers [64].

The Young’s modulus of the nucleus varies across cell types and experimental techniques. However, it is generally accepted that for cells in culture, the nuclear stiffness ranges from ~0.1 to 10 kPa, which is ~2 to 10-fold higher than that of the cytoplasm [65–69]. Use of AFM confirmed that NE is stiffer than the cell membrane in chondrocytes within intact cartilage tissue cultured ex vivo [70]. Importantly, degradation of cartilage ECM by metalloproteinases reduced matrix, cell and nuclear stiffness, suggesting that the tissue microenvironment governs the mechanical properties of cells [70]. However, assessing nuclear and cell stiffness in the cell’s native environment remains a formidable challenge, primarily due to the inherent limitations of conventional devices like AFM or micropipette aspiration, which require direct contact with cells. The advent of non-invasive methods, such as Brillouin microscopy [12,71,72], has the potential to enhance our understanding of the mechanical properties of cells in vivo.

Besides nuclear rigidity, nuclear volume expansion may also hinder confined cell migration by shrinking the space between the nucleus and the channel walls, thereby elevating the viscous drag on the plasma membrane, which in turn decreases migration velocity [73]. The role of lamins in volume regulation is less evident in part due to the pleiotropic consequences of their experimental manipulation. Depletion of lamin-A in MEFs caused an increase in nuclear volume [35]. However, the reconstitution of lamin-A failed to rescue the increase in nuclear volume. In contrast, depletion of lamin-B1 strongly reduced nuclear and cell volume, and both changes were rescued by lamin-B1 re-expression. Moreover, the various lamin-manipulated MEF cell lines showed significantly different correlations between the nuclear and cell volumes [35], suggesting that altered lamin levels perturbed the mechanisms maintaining cell type-specific nuclear-cellular volume ratios [74]. These results are consistent with the evidence for the multifactorial control of nuclear size [75], including the critical, albeit likely, cell type-specific role of nuclear-cytoplasmic transport receptors [75,76] and perinuclear ER in developing embryos [77].

In addition to their roles in regulating mechanical properties, nuclear lamins contribute to nuclear mechanosensing pathways [78,79]. Effective force transmission from the cell surface to the nucleus through the cytoskeleton is achieved through the LINC complex [41] (Fig. 1A). The LINC complex spans the nuclear membrane through the interaction between C-terminal KASH (Klarsicht, ANC-1, Syne homology) domain proteins known as nesprins and SUN (Sad1 Unc-84) domain proteins [80]. Nesprins extend from the outer NE and interact directly with some of the cytoplasmic intermediate filaments or cytoskeleton components (actin, tubulin), whereas SUN proteins localized in the inner NE anchor the LINC complex to lamins (Fig. 1A) [80–82]. Direct interactions of lamins with the LADs of the chromatin [83] complete the LINC-mediated transmission of mechanical forces from the cell’s exterior through the cytoplasmic cytoskeleton and intermediate filaments to the genome [82]. The intranuclear organization of the genome, particularly the typically perinuclear accumulation of transcriptionally silent heterochromatin, has been proposed to affect nuclear deformability and enhance nuclear sturdiness [84]. In particular, the lamin-A-dependent accumulation of heterochromatin at the nuclear periphery through LADs (Fig. 1B) [83] might increase overall nuclear stiffness and could mediate the LINC-dependent mechanical responses of chromatin (Fig. 1A) [83]. However, it remains unclear if the LAD-mediated chromatin response depends strictly on LINC-mechanotransduction or involves contribution of MIC-dependent signaling [85].

Applying force to nesprin-1 induces lamin-A/C recruitment to the LINC complex (Fig. 1A) and nuclear stiffening mediated by Src family kinase phosphorylation of the LEM-domain protein emerin [86]. Subsequent studies performed on myoblasts revealed that nesprin-1 and lamin-A are required for cells to alter stress fiber accumulation, focal adhesion formation, and traction forces in response to the rigidity of their matrix [87]. Interestingly, studies in human subjects revealed that exercise-associated mechanical forces induce lamin A recruitment to myonuclei, promoting nuclear stiffening and spherical morphology independently of age [88]. Tissue stiffening can alter lamin-A/C phosphorylation states [89]. Culturing mesenchymal stem cells on soft gels favored increased phosphorylation-mediated degradation of lamin-A/C, resulting in softer nuclei and consequent reduction in myosin IIA activity [89]. The use of Brillouin microscopy, an all-optical technique for measuring nuclear mechanical properties [71,72], suggests that the nucleus modulates its nuclear stiffness in response to distinct confinement geometries [12]. Vertical compression exerted along the dorsoventral polarity axis of the cells triggers perinuclear myosin II-dependent nuclear stiffening, which may be regulated through phosphorylation of lamin-A/C, as disruption of perinuclear myosin significantly increased lamin-A/C phosphorylation [12]. In contrast, the absence of perinuclear myosin II in cells subjected to lateral compression directed perpendicularly to the dorsoventral polarity axis is consistent with the lack of nuclear stiffness alteration [12]. Taken altogether, feedback between the nucleus and extracellular forces can tune internal cell stiffness to match the local microenvironment.

Physical signals regulate chromatin organization, nuclear rigidity and gene expression

In addition to the lamina, the presence of the genomic DNA and the state of its activity constitute major determinants of the mechanical properties of the nucleus at various length scales, including the nucleus as a whole [85,90]. Thus, nuclear rigidity, morphology, and mechanosensing pathways can be regulated by the histone modification state of chromatin [85,91]. Condensed heterochromatin promotes stiffer nuclei with an ellipsoidal morphology, whereas loose, transcriptionally active euchromatin promotes softer nuclei that form nuclear blebs independently of changing lamina content [91] (Fig. 1D). Physical cues can restructure and reorganize chromatin [41]. Heterochromatin is often located at the nuclear periphery through direct interactions of LADs with lamins A and B [92], often promoting gene silencing [41,85,93]. Its repositioning to the nuclear interior is associated with transcriptional activation in specific contexts [41,94]. However, the relationship between its radial position and transcriptional activity is complex, partly owing to the diverse responsiveness of various gene promoters to the LAD environment [92].

Chromatin exhibits mechanosensitive responses to force application [79,95–97] that could be adaptive in a cell type- or context-specific manner. Specifically, nuclear deformation by mechanical stretch in non-transformed epithelial cells triggered calcium release from the endoplasmic reticulum (ER) to reduce H3K9me3 heterochromatin, resulting in nuclear softening and protecting genomic integrity, which is consistent with the remarkable mechanical stress resistance of normal epithelial tissues [96]. In contrast, migration of HT-1080 fibrosarcoma cells through stiff, polydimethylsiloxane (PDMS)-based microchannels induced heterochromatin formation as the degree of confinement increased, thereby leading to global repression of transcriptional activity [95]. Although increases in global heterochromatin levels were not detected in cells migrating through narrow pores in 3D collagen matrices, Assay for Transposase-Accessible Chromatin (ATAC)-seq analysis identified reduced chromatin accessibility near centromeres and telomeres, consistent with heterochromatin formation [95]. Interestingly, increased chromatin accessibility was noted at genes associated with chromatin silencing, tumor invasion and DNA damage response (DDR) [95]. Consistent with these findings, nuclear compression in the dorsoventral direction sufficient to induce lamina breakage affects the expression of genes involved in regulating DDR, DNA metabolism and nucleolar RNA production [98]. Application of stress on integrins via 3D magnetic twisting cytometry is propagated through the actin cytoskeleton to the LINC complex via SUN1/2 and then through lamina-chromatin interactions to induce chromatin stretching and gene transcription [97]. Thus, forces applied to cells are sensed by the nucleus, resulting in long-term changes through regulation of gene expression. Among the questions open for future exploration is whether the likely feedback between the physical properties of the nucleus as a whole versus chromatin organization/activity is universal or cell type-specific, reflecting different adaptations in normal versus cancer cells.

Phase separation in nuclear mechanosensation

Liquid-liquid phase separation (LLPS) results from the tendency of macromolecules, such as proteins, DNA and RNA, to spontaneously segregate or demix from a homogeneous solution into distinct phases, due to the preferred homotypic interactions (Fig. 1E) [99–101]. Phase separation contributes to the chromosome dynamics and genome organization, including the behavior of chromatin as a polymer fiber, and the formation of euchromatin and heterochromatin nuclear regions [40,102]. Softer, euchromatin-rich regions of the nucleus favor the formation of membraneless nuclear condensates, including Cajal bodies, paraspeckles, promyelocytic leukemia (PML) nuclear bodies and nucleoli [100,101]. As the condensate grows, chromatin is mechanically displaced, resulting in genome restructuring. Interestingly, nuclear condensates (paraspeckles) are also formed at the nuclear front during migration through moderately, but not tightly, confining microchannels [103]. Although the role of nuclear bodies in confined migration remains unclear, recent findings reveal a positive correlation between paraspeckle formation and cell migration velocity [103].

The application of dynamic external forces via integrins induced rapid dissociation of proteins comprising Cajal bodies (Fig. 1E), potentially affecting their role in gene expression regulation [104]. Similarly, studies involving keratinocyte adhesion on micropatterned surfaces revealed that cell adhesion to ECM modulates the organization of the largest subnuclear organelle, the nucleolus, affecting ribosomal transcription and ribogenesis [105]. Recent studies showed that relatively short (2–10 min) integrin-mediated chromatin compression induced prolonged (tens of minutes) elevation of HP1 heterochromatin protein diffusivity in nuclei, likely via “fluidizing” the heterochromating condensates, thus enabling persistent mechanomemory [106]. While essential questions on the underlying mechanisms remain to be addressed [100], these studies indicate that LLPS likely contributes to many aspects of nuclear mechanosensation.

Nuclear Integrity, DNA damage, nuclear adaptation, and cancer progression

The mechanical stress exerted on the nucleus during confined cell migration can disrupt nuclear integrity and induce genomic instability [62,73,107,108]. Seminal studies by the Discher, Lammerding, and Piel groups have established that cells migrating through narrow constrictions can experience transient NE ruptures, characterized by the rapid exchange of material between the nucleus and the cytoplasm [62,107,108]. Nuclear rupture events occur at sites of high curvature [109] and coincide with or follow the formation of NE blebs, which are spherical protrusions of the nuclear membrane devoid of lamin B1 and nuclear pores [107]. Actomyosin contractility can induce NE blebbing and rupture through different mechanisms: a) physically squeezing the nucleus’s apical and basal surfaces [107,110], b) pulling the nucleus [111] and/or c) pushing it [73]. The latter mechanism is prevalent during migration through stiff, confining microchannels. The elevated pressure at the cell posterior may induce NPC stretching at the nuclear trailing edge, resulting in elevated nuclear influx and retention of diffusible cytoplasmic constituents and water, which in turn pressurizes the nucleus, causing nuclear expansion and anterior NE blebbing and rupture [73] (Fig. 1F,G). Interestingly, nuclear export inhibition also promoted nuclear bleb formation, underscoring the importance of nuclear flux homeostasis, including the active macromolecular transport, in nuclear integrity [73].

Nuclear rupture events lead to DNA damage, as indicated by the presence of γH2AX and 53BP1 foci, markers of DNA double-strand (ds) breaks [62,107]. Furthermore, changes in chromosome copy number have been observed following NE rupture due to constricted migration [108]. Nuclear rupture elicits these detrimental effects by inducing the translocation of cytoplasmic exonuclease TREX1 into the nucleus, leading to DNA damage [112] (Fig. 1G). TREX1 depletion suppresses tumor invasion, indicating TREX1 as a potential therapeutic target to halt metastatic spread [112]. Because TREX1 acts on exposed 3’ dsDNA strands [113], it remains unclear if previous confinement-induced DNA breaks are required for the TREX1-amplified DNA damage. NE rupture also promotes the persistent mislocalization of the DDR factors Ku70, Ku80, and BRCA1 to the cytoplasm, reducing the rate of DNA repair and exacerbating the damage [108,109]. NE rupture readily exposes cytoplasm to nuclear dsDNA, which is sensed as a cellular threat through the innate immune response cGAS/STING (cyclic GMP-AMP synthase pathway/Stimulator of interferon genes) pathway. The cGAS/STING response is particularly relevant to genomically unstable cells containing micronuclei that possess a fragile NE and are susceptible to massive genomic rearrangement called chymotrypsis [114,115] (Fig. 1G). It remains unclear whether other factors can enter or exit the nucleus during nuclear rupture and how such events influence tumor cell behavior.

Confinement can also trigger genetic changes and DNA damage without NE ruptures. Mild cell compression during mitosis promotes chromosome loss, which is further exacerbated in myosin-IIA-depleted cells, suggesting that myosin II protects against force-induced genetic alterations [116]. Confinement-induced nuclear ruptures are infrequent in certain cell lines, such as breast cancer MDA-MB-231 cells [117]. In these cells, nuclear squeezing increases replication stress and promotes DNA damage at replication forks. Consistent with these findings, confined migration has been shown to reduce cell division [21,111,118–120]. Confinement hinders nuclear size increase following G₁ cell cycle phase, resulting in reduced proliferation and abnormal divisions [118]. The reduced frequency of cell division persists even after the cells escape from confining spaces, suggesting a memory effect. Cell treatment with the antioxidant Glutathione Monoethyl Ester (GSH) and the myosin II inhibitor blebbistatin suppresses confinement-induced DNA damage and rectifies cell cycle abnormalities [111]. Similar results are also obtained by overexpressing three DDR factors (KU70, KU80, and BRCA1) and simultaneously treating cells with GSH [111].

Although cell migration through confining microenvironments can cause genomic instability, it rarely promotes cell death [62,107,108]. These observations suggest that cells develop compensatory mechanisms to adjust to confinement, possibly through genomic instability-driven selection advantage [121]. Recent work has shown that long-term cell entrapment in microchannels that deform the cell and its nucleus can trigger p53-dependent cell apoptosis [21]. However, a significant fraction of confined cells survives because the mechanotransducer Yes-associated protein (YAP) exits the nucleus to reduce the nuclear rupture frequency, resulting in downregulation of p53 activity [21]. These findings underscore the critical role of YAP as a key mediator of mechanoadaptation. Nuclear volume also adapts to prolonged confinement. Short (2 h) cell exposure to uniaxial confinement has been reported to promote NE unfolding, and nuclear spreading, while maintaining the nuclear volume relatively unchanged [122]. However, after the first division, confined, but not unconfined, cells display reduced NE tension and volume, suggesting that mitosis allows cells to establish a new homeostatic state. This nuclear adaptation requires activation of myosin II contractility, consistent with its pivotal role in mitosis.

Emerging evidence suggests that confinement-induced nuclear deformation and DNA damage exacerbate tumor invasion and metastasis. Invasive cells located at the periphery of breast tumors exhibit squeezed nuclei, signs of NE rupture and pronounced DNA damage due to TREX nuclear influx [112]. The inner NE protein lamin-associated polypeptide 1 (LAP1), which promotes nuclear blebbing, is highly upregulated at the invasive front of melanomas and in metastatic lesions [123]. Importantly, out of the two LAP1 isoforms, only the short isorform LAP1C promotes nuclear blebbing, confined cell migration and invasion [123]. In addition to promoting nuclear deformation, rupture and DNA damage, confinement can confer a survival advantage to metastasizing tumor cells by inducing cell resistance to anoikis and chemotherapeutic treatments [124,125].

Mechanical regulation of nucleocytoplasmic transport

Macromolecules transport between the nucleus and the cytoplasm through NPC channels (Fig. 1C) via passive or energy-dependent, facilitated diffusion [126,127]. A hydrophobic meshwork of disordered phenylalanine-glycine (FG)-rich domains of nucleoporins (Nups) lines the inner walls of NPC channels, limiting but not entirely blocking the concentration gradient-driven passive diffusion of cargos larger than 30–60kDa [128]. Nuclear transport receptors (NTRs, importins, and exportins) engage in multiple low-affinity interactions with the FG repeats, enabling rapid transitions through NPC channels while either alone or bound to their cargos through their nuclear localization signal (NLS) or nuclear export signal (NES) motifs. The driving force of the active NTR-mediated transport is the large nuclear-cytoplasmic concentration gradient of the GTP-bound form of the small nuclear GTPase Ran, resulting from the nuclear localization of Ran GEF (RCC1) and cytoplasmic residence of the RanGTPase activating protein RanGAP1 and its cofactors RanBP1,2. RanGTP binding to exportins enables their NES cargo loading in the nucleus. Upon transition of the trimeric export complex to the cytoplasm, GTP-to-GDP hydrolysis on Ran, catalyzed by RanGAP1/ RanBP1/2, dissociates the complex, releasing the NES cargo. Conversely, importins load their cargos in the cytoplasm, diffuse through NPC FG repeats to the nucleus, where RanGTP binding to the importin dissociates the NLS cargo [129]. In addition to the NTR-facilitated transport, several other non-canonical mechanisms mediating nuclear-cytoplasmic protein shuttling were identified (reviewed in [130]). While exportin 1, 5 and exportint enable active nuclear export of several classes of RNAs [131,132], the majority of nuclear mRNA export relies on the NXF1/NXT1 heterodimeric receptor and involves the active contribution of many RNA binding proteins and several Nups [133].

As one of the largest cellular protein complexes, NPCs can undergo conformational changes in response to NE tension, affecting their molecular gating function. Hypertonic shocks or energy depletion, which reduce NE tension, lead to NPC constriction, resulting in diminished passive GFP transport [134]. In contrast, increased substrate stiffness induces actomyosin-dependent nuclear flattening, which stretches the NE and NPCs, thus enhancing the passive diffusion through the widened NPC channels [45,135]. NPC stretching-induced nucleocytoplasmic shuttling resulted in nuclear influx of key transcription regulators, such as YAP (65 kDa) [45], SMAD3 (48 kDa), Twist1 (21 kDa) and Snail (29 kDa), and to a much smaller extent, NF-κB [135], indicating that the mechanosensitive NPC dilation can affect cell behavior through extensive transcriptional changes. Detailed experimental and computational analysis revealed complex consequences of NPC dilation on passive versus active NCT [135]. The mechanosensitivity of passive nuclear import of an engineered GFP cargo sharply decreased with molecular mass ≥54 kDa, and the predicted mechanosensitivity of active nuclear import was affected by a balance of molecular mass and the NLS strength. Additional factors, including cargo surface charge [136] and mechanical properties [137], could affect their mechanosensitivity. Among the questions awaiting further investigation are the causes of the observed much lower mechanosensitivity of nuclear export compared to nuclear import [135].

The impact of 3D confinement on NCT remains largely unexplored. While elevated stiffness and nuclear flattening facilitate the translocation of cytoplasmic YAP to the nucleus, tight confinement triggers YAP nuclear exit, in the absence of nuclear rupture, and in a partially Exportin 1-dependent manner [21]. Confinement-induced NCT of the RhoA regulators Ect2 (GEF) and RacGAP1 (Rho GAP) was shown to underlie leader cell dissociation from collectively migrating tumor cells [138]. While both RacGAP1 and Ect2 are typically nuclear during interphase, RacGAP1 relocates to the central spindle during cytokinesis and recruits Ect2 to locally activate RhoA, enabling its critical role in actomyosin contractile ring assembly. Collective cell migration in confinement leads to cytosolic enrichment of both Ect2 and RacGAP1, which contributes to RhoA-mediated leader cell dissociation [138]. Intriguingly, while Ect2 further accumulates in the cytoplasm after leader cell detachment, RacGAP1 starts returning to the nucleus about 50 min before dissociation [138]. While the underlying mechanism remains to be elucidated, these results suggest the potential role of RacGAP1 nuclear import in sensing the leader cell commitment to dissociate from collective strands.

CROSSTALK BETWEEN MECHANOSENSATION BY THE NUCLEUS AND TRANSMEMBRANE CHANNELS IN CELL MIGRATION

In addition to the nucleus, several classes of transmembrane MICs have been identified as crucial mechanosensing regulators of cell migration. The crosstalk between the nuclear and MIC-dependent mechanosensing responses controls cell migration through complex in vivo 3D environments.

The role of calcium signaling and MICs in cell migration

Calcium is a critical second messenger that interacts with numerous proteins involved in regulating actin, focal adhesions, and myosin II to modulate actomyosin contractility and migration [139]. As a ~10,000-fold gradient of free calcium ions exists across the plasma or ER membranes relative to the cytoplasm, calcium mobilization can initiate a wide range of signaling pathways within the cell [139]. As such, the amplitude and spatiotemporal characteristics of calcium signaling events are essential regulators of the cell migration machinery [140].

During cell migration in vivo, cells are exposed to different cues (e.g., compression, hydraulic pressure, extracellular fluid viscosity, fluid flow, substrate viscoelasticity, pH and osmolarity changes etc.), which can alter plasma membrane tension [16,141]. MICs constitute a family of pore-forming proteins localized at the endoplasmic reticulum [142], nuclear [143] and plasma membranes [27]. MICs can sense and respond, likely but not exclusively, to changes in membrane tension via a conformational change from a closed to open state. An alternative mechanism involves their interaction with the cytoskeleton, the ECM, or both. However, these two models may not be mutually exclusive, as reviewed in [144]. MIC activity can also be modulated by the membrane lipid composition and order (e.g., in lipid rafts or other membrane lipid subdomains) or changes in intracellular mechanically-generated messengers [144].

MICs, such as those in the Transient Receptor Potential (TRP) and Piezo families, facilitate calcium influx via a mechanism known as channel gating [27], which refers to channel opening triggered by mechanical stimuli. Calcium influx through MICs regulates cell migration by altering focal adhesion dynamics via activation of calpain, a cysteine protease that cleaves various adhesion components, such as focal adhesion kinase (FAK) [27,145], and by modulating cell contractility [15]. One of the best studied MICs of the TRP superfamily is the TRP cation channel subfamily M member 7, also known as TPRM7. TPRM7 is involved in directional persistence, actin assembly, lamellipodia formation, focal adhesion dynamics, and myosin IIA filament stability [146–148]. TRPM7 is activated by cell swelling [149], shear stress [17], and hydraulic pressure [16,29]. By acting as a sensor of hydraulic pressure, TRPM7 bestows tumor cells with the ability to identify the path of least resistance during migration in confinement [29]. Specifically, TRPM7 activation due to elevated hydraulic pressure mediates calcium influx that supports the formation of a thick cortical actin meshwork with an elevated density of myosin IIA motors, which propels cells into channels of lower resistance [29], a phenomenon known as barotaxis [150]. On the other hand, by acting as a fluid shear sensor, TRPM7 suppresses cell intravasation by activating RhoA-myosin II contractility at the cell edge encountering shear flow, which in turn drives the reversal of migration direction via a calmodulin/IQGAP1/Cdc2 pathway [17]. In line with this notion, normal fibroblasts, which display a higher TRPM7 expression than fibrosarcoma cells, fail to intravasate. Moreover, overexpression of TRPM7 in fibrosarcoma cells confers fluid shear sensitivity and suppresses intravasation in vitro and in vivo [17].

Many other members of the TRP superfamily of MICs also play important roles in cell motility. For instance, the TRP channel subfamily V member 4 (TRPV4) responds to mechano-osmotic stimuli and regulates motility by activating calpain at focal adhesion sites, which promotes focal adhesion disassembly and rear-end retraction [151]. Although TRPs can be activated by both physical and chemical cues, Piezo 1/2 are primarily gated by mechanical signals [27,152]. Physical confinement triggers calcium influx through Piezo 1 during Chinese Hamster Ovary (CHO) or melanoma cell entry into fibronectin-coated microchannels [153]. This elevated intracellular calcium level, in synergy with myosin II, suppresses the activity of cyclic adenosine monophosphate-dependent protein kinase A (PKA) and promotes efficient migration in confinement [153]. Similarly, brain metastatic cells from breast cancer (MDA-MB-231) migrate efficiently through confining spaces via Piezo 2-mediated calcium influx, which activates RhoA and controls stress fiber and focal adhesion formation [154]. The crosstalk between Piezo 2 and RhoA activation involves the recruitment of Fyn kinase and calpain activation at the leading edge of cells migrating in confinement [154]. It is well established that physical confinement induces a mesenchymal to amoeboid transition [7,8,12,73]. Interestingly, upon application of uniaxial compression, Dictyostelium cells also switch from pseudopodial to bleb-based migration by sensing pressure via Piezo channels [155]. Collectively, these findings highlight the crucial role of MICs in controlling the modes and mechanisms of confined cell migration.

A major difference between distinct MICs is their sensitivity to the strength of mechanical stimuli and their specific interactions with different partners in their signaling microdomains generated beneath the plasma membrane, as Ca²⁺ signals cannot travel far away, typically a few micrometers, in the very crowded intracellular environment. Along these lines, mechanical sensing of physiologic levels of strain in chondrocytes is mediated by TRPV4, whereas that of supraphysiological strains (>45%) is via Piezo1/2 [156].

The interplay between MICs, ion transporters, and ion channels in cell migration

In addition to the role of MICs in preserving ion gradients across the plasma membrane and enabling select ions to act as secondary messengers, the activity of MICs, ion transporters and ion channels regulates cytoplasmic ion concentration and thus the osmotic pressure of the cell [157]. The osmotic pressure, along with the hydrostatic or hydraulic pressure, constitute the overall intracellular pressure [157]. At equilibrium, osmotic and hydraulic pressures are equal, and cell shape and volume remain constant [157]. However, an imbalance between the hydraulic and osmotic pressures results in water flow across the plasma membrane, which can alter cell shape and volume and initiate cell movement [157]. This is the underlying principle for the osmotic engine model of cell migration, where cells use directed water permeation to propel in confinement even after complete actin disruption [30]. Specifically, a cell migrating in a confining channel establishes a spatial gradient of ion transporters/channels and aquaporins (AQPs) in the plasma membrane so that local swelling at the leading edge and shrinkage at the trailing edge, respectively, facilitate net cell forward movement [30]. The Na⁺/H⁺ exchanger 1 (NHE1), which polarizes along with AQP5 to the cell leading edge, promotes isosmotic cell swelling consistent with its role in regulatory volume increase [28,30] (Fig. 2A). On the other hand, the SWELL1 (LRRC8A) chloride channel, which is preferentially enriched at the cell trailing edge along with AQP4, mediates cell shrinkage and regulatory volume decrease [28] (Fig. 2A). This polarization pattern results in a net water inflow at the cell front and expulsion at the cell rear, propelling cell migration and driving breast cancer cell extravasation and metastasis [28]. These findings are in agreement with recent data obtained using primary human neutrophils, which highlight the importance of water uptake and NHE1-mediated cell swelling in rapid neutrophil migration following chemoattractant stimulation [158]. The role of water uptake, albeit via macropinocytosis, has also been demonstrated for immature dendric cells, rendering them insensitive to external hydraulic resistance, thereby allowing them to more exhaustively explore their surrounding microenvironment [150]. In contrast, mature dendric cells downregulate micropinocytosis, and as such, choose the path of least resistance. In sum, these studies illustrate the interplay of osmotic and hydraulic pressure, and water permeation in propelling cell locomotion.

Figure 2. Ion fluxes and nuclear signaling facilitate cell migration through confining spaces.

(A) Cells in confinement polarize distinct ion transporters, ion channels, and aquaporins at the cell front and rear. NHE1 and AQP5 are preferentially enriched at the cell leading edge, promoting cell swelling, which in turn increases membrane tension, triggering TRPV4-dependent calcium influx and activation of actomyosin contractility. At the cell trailing edge, enrichment of the SWELL1 chloride channel and AQP4 induces cell shrinkage. Moreover, compressive forces due to confinement in the dorsoventral axis stretch the NE, stimulating cortical actomyosin contractility through a mechanism that involves calcium release from internal stores, cPLA2 activation, and arachidonic acid production. (B) Functioning like a piston, the nucleus pressurizes the cell front, activating TRPV4 and NHE1, which mediate the influx of calcium and sodium ions, thereby promoting protrusion expansion and cell migration through confining viscoelastic gels.

External hydraulic resistance can also vary due to changes in the extracellular fluid viscosity. Although most migration studies have been performed in medium with a viscosity close to that of water (0.7 cP at 37°C), the viscosity of the interstitial fluid can reach up to 3.5 cP, and can be further increased in certain pathological conditions, including cancer [159]. Elevated, albeit physiologically relevant, levels of viscosity counterintuitively increase cell migration in 2D, 3D, and in confinement as well as cell dissemination from 3D tumor spheroids. Elevated viscosity causes mechanical loading at the cell leading edge, which induces Arp2/3-mediated actin remodeling that in turn enhances NHE1 polarization to the cell leading edge via binding to its partner ezrin [15]. NHE1 increases cell volume via water uptake, which leads to increased membrane tension that triggers the activation of MIC TRPV4 to mediate calcium entry, and ultimately promote RhoA/ROCK/myosin II contractility, migration and metastasis [15] (Fig. 2A).

The crosstalk between nucleus and MICs, ion transporters, and calcium signaling

The nucleus is an essential regulator of compartmentalized intracellular pressure [73,160]. This was first demonstrated by Petrie et al., who discovered that the nucleus can act as a piston, which is pulled towards the cell front to selectively pressurize the cell anterior compartment, driving lobopodial migration in 3D ECM environments [160]. The lobopodial mode of migration, which is characterized by asymmetric intracellular pressure and a large blunt cylindrical protrusion or lobopodia at the cell leading edge, is observed in highly crosslinked, linearly elastic 3D matrices that support high adhesion and Rho/ROCK/myosin II contractility [160] (Fig. 2B). The nuclear piston model, originally identified in fibroblasts [160], was also present in fibrosarcoma cells migrating in linear elastic 3D ECM microenvironments only when their matrix metalloproteinase activity was inhibited [161]. According to the nuclear piston model, the nucleus is pulled forward through the coordinated action of actomyosin, vimentin, and nesprin 3 to pressurize the cell anterior [160]. This nuclear-dependent pressurization of cell protrusions was recently reported to activate TRPV4 and NHE1 in mesenchymal stem cells, elevating calcium and sodium, respectively, within these protrusions (Fig. 2B). This influx of ions allows osmotic pressure to outcompete hydrostatic pressure within the protrusion, resulting in water influx, protrusion expansion, and cell migration through confining viscoelastic gels [162].

While MICs primarily reside on the plasma membrane, the nuclear membrane also contains stretch-activated calcium-permeable channels [163], including Piezo channels [143]. Stretch-activated calcium channels on the nuclear and ER membranes play a critical mechanosensory role in confinement [164,165]. Moderate cell compression along their dorsoventral axis stretches the NE, resulting in elevated nuclear membrane tension and calcium release from internal stores, which ultimately elevates myosin II contractility and facilitates confined cell migration [164] (Fig. 2A). Chelation of extracellular calcium or inhibition of plasma membrane-associated stretch-sensitive channels had no effects on the contractile response in confinement, suggesting that the lack of integrin or plasma membrane engagement in this process. In contrast, intracellular calcium, intracellular stretch-activated calcium channels and the nuclear tension sensor cPLA2 or its downstream byproduct arachidonic acid are required for inducing contractility in confinement, suggesting that this process is activated from a signal emanating from the perinuclear ER and/or the NE [164]. These studies highlight that a crosstalk between nuclear and calcium-dependent signaling is essential for cells’ adaptive responses during confined migration.

CONCLUDING REMARKS AND PERSPECTIVE

Migrating cells employ a wide repertoire of mechanosensors to discern and decode the physical and topographical features of their local microenvironment. The nucleus and MICs lie at the core of this mechanosensing process, mediating transient or lasting cellular responses that shape the cell’s migratory behavior and fate. However, the detailed mechanisms underlying the interplay between the nucleus and MICs remain largely unexplored. Recent work using immune cells to study amoeboid migration through mazes has demonstrated that the front-facing nucleus serves as a mechanical gauge, allowing cells to choose the migratory path of least hydraulic resistance [166]. The microtubule organizing center (MTOC), which is typically positioned behind the nucleus in these cells, acts as the spatially associated directional selector responsible for the retraction of cytoplasmic protrusions that may still be lingering in pores of higher hydraulic resistance via regulation of microtubule dynamics [166]. As TRPM7 also influences decision-making strategies in epithelial cells [29], exploring the potential crosstalk between the nucleus and TRPM7 in the context of sensing hydraulic resistance and possibly other physical cues deserves more attention. The observation that Piezo1 localizes to the NE [143] raises questions regarding its potential interaction with NE proteins. Specifically, could mechanical force-dependent activation of Piezo channels on the NE impact NPC permeability, NCT, and consequently other biophysical nuclear features, such as stiffness? Conversely, could the structural components of the nucleus control the localization and activity of the Piezo channels? While NE stretching leads to Ca²⁺-dependent activation of contractility [164], the consequences of NE ruptures on MIC activity, Ca²⁺ signaling and the migration machinery remain largely unknown. Addressing these gaps in knowledge could provide important insights into the mechanisms coordinating nuclear and MIC signaling in the regulation of cell migration behaviors in confinement.

In addition to mechanistic studies, the clinical relevance of nuclear and MIC signaling during confined cell migration merits further exploration. Several studies suggest that altered expression of nuclear lamins or MICs [27] is characteristic of a variety of cancers and associated with a malignant phenotype [48,64]. These molecules, however, can either facilitate or hinder the metastatic process, contingent upon the specific stage of metastasis. For example, while diminished lamin A levels can promote invasion [48], they may compromise the survival of tumor cells in circulation [167]. Similarly, elevated TRPM7 levels, while enhancing cancer cell migration [168], could inhibit intravasation by rendering cells more sensitive to shear stress [17]. Thus, devising interventions tailored to specific metastatic stages could prove more efficacious in impeding metastasis.

Despite recent progress in understanding the molecular hallmarks of cancer, the presence of changes in nuclear size, shape, and organization remains one of the most reliable identifiers of cancerous tissues [169]. However, the relationship between these morphological changes and a malignant phenotype is still incompletely understood (see also Outstanding questions). Furthermore, physical deformation of the nucleus of metastatic cells may promote changes in gene expression, NE rupture and genome instability, driving cancer progression [62,107,108]. Going forward, combining mechanistic insights about nuclear and MIC signaling during 3D migration with cancer hallmarks may lead to the development of novel therapeutic targets for cancer metastasis.

Outstanding questions.

• Many proteins critically involved in regulating mitotic cell division are sequestered in interphase nuclei. Since late S/G₂ nuclei contain nearly double DNA content compared to G₀/G₁ nuclei, does confined migration affect actively dividing cells differently from post-mitotic, non-dividing cells? Does the premature cytoplasmic release of mitotic factors contribute to the metastatic migratory cell phenotypes?

• Although numerous mechanical force-induced changes in gene expression have been documented, the precise mechanisms and consequences of confined migration on RNA metabolism remain to be elucidated. What are the effects of confinement on pre-mRNA splicing, mRNA nuclear export, and cytoplasmic translation? Single-cell RNAseq, special single cell ptoteomics and associated methods could provide crucial insights into the impact of confined migration on gene expression.

• How do symmetric versus asymmetric 3D topographies and physical forces affect nuclear compartmentalization and active/passive NCT in migrating cells? Is this transport affected globally involving all NPCs, or are there asymmetric, spatially localized changes affecting NPCs at the walls or at cell rear/front?

• What is the role of confinement-induced changes in NCT and MIC-mediated signaling in cell migratory behavior and mechanomemory? How does the interplay between the nuclear and MIC-mediated mechanoresponses affect collective migration and leader cell dissociation?

• How does the confined migration affect LLPS -mediated nuclear processes, such as formation and function of nuclear bodies? Do LLPS-mediated processes (including changes in chromatin structure/accessibility) contribute to mechanomemory? What are the consequences of the formation of nuclear condensates in the short- and long-term migratory behavior of cells?

• Besides Piezo 1, do other MICs localize at the nuclear membrane? Can MIC-dependent signaling affect nuclear mechanoresponses, such as chromatin modification, NPC permeability, or nucleocytoplasmic shuttling?

• What is the clinical relevance of the crosstalk between nuclear and MIC signaling? Could it provide new avenues for targeting metastatic cells? How do they impact different stages of the metastatic cascade?

HIGHLIGHTS.

• The nucleus is a key cell mechanosensor of the local microenvironment.

• Non-destructive nuclear mechanosensation responses in confinement involve the buildup of the nuclear lamina, nuclear pore complex-stretching-dependent exchange of nuclear-cytoplasmic constituents and nuclear pressurization.

• Destructive nuclear mechanoresponses include the nuclear depletion of DNA repair factors and nuclear influx of TREX1 exonuclease, which promote DNA damage, as well as the cytoplasmic access of dsDNA that can trigger cGAS/STING response.

• Ion channels are among the first molecules to sense and respond to different physical stimuli, including confinement, by regulating intracellular ionic concentrations and water fluxes, thereby altering cell motility.

• The crosstalk between nuclear mechanotransduction and ion channel-mediated signaling enables efficient cell migration through confining spaces.