Mechanobiology of the Nucleolus

ABSTRACT

Ribosome biogenesis is an essential and energy‐demanding process required to maintain cellular proteostasis. The nucleolus, which orchestrates this vital process, assembles as a nuclear condensate and exhibits remarkable plasticity in its structure, function, and protein composition. This dynamic membraneless organization of the nucleolus contributes to its involvement in diverse signaling pathways, influencing cell cycle regulation, proliferation, apoptosis, differentiation, and cellular stress response. Here, we focus on how mechanical cues influence nucleolar biology. Although classical mechanotransduction is mediated by membrane‐anchored signaling proteins and the cytoskeleton that propagate forces within the cell, how mechanical cues influence the organization and function of dynamically assembled, membraneless condensates remains an open question. In this review, we explore how mechanical stimuli impact the nucleolus, the most prominent nuclear condensate. We examine how extrinsic forces alter nuclear structure, thereby affecting nucleolar organization, and function. Finally, we highlight emerging evidence that positions the nucleolus as a key mediator of nuclear mechano‐adaptation, linking extracellular matrix (ECM) cues, migration, confinement, and external mechanical stress to nucleolar assembly and ribosome biogenesis.

Mechanical forces reshape nuclear structure and reorganize the nucleolus, altering its role in ribosome biogenesis. This review examines how mechanical cues impact nucleolar assembly and function. We propose that the nucleolus acts as a mechano‐responsive condensate integrating mechanical stress with cellular homeostasis.

1. Introduction

Modern cell biology has been largely dominated by biochemical and molecular signaling paradigms that shape cell and tissue behavior. However, evidence of the role of mechanical forces in tissue development emerged as early as the late 19th and early 20th century. Seminal studies by Wolff, Fell, and Canti provided initial empirical evidence that mechanical stimuli impact tissue morphogenesis and development (Fell and Canti 1934; Wolff 1892). Modern mechanobiology emerged in the late 20th century with the advent of atomic force microscopy (AFM), optical tweezers, and advanced imaging modalities. These technologies enabled controlled perturbation of cell and tissue mechanical environments coupled with high‐throughput imaging, providing comprehensive approaches to decode cellular biomechanics and the role of mechanical cues in cell functioning and tissue development.

Building on these historical foundations, mechanobiology now aims to decipher the intricate mechanisms through which cells sense, respond, and adapt to their mechanical environment. Studies on cell‐ECM interactions have uncovered a complex network of feedback mechanisms wherein cells and the ECM are able to dynamically sense and modulate each other's mechanical properties to maintain tissue homeostasis (Hall et al. 2016; Harris et al. 1981; Winer et al. 2009). Moreover, changes in cell density, ECM availability, and matrix stiffness impose mechanical constraints on the cells, triggering downstream signaling responses (Natarajan et al. 2015; Zarkoob et al. 2015). The conversion of these extrinsic mechanical cues into intracellular biochemical signals, via membrane receptors and cytoskeletal elements, delineates the classical pathways of mechanotransduction. These pathways play a critical role in regulating cell fate and function, influencing major cellular events such as cell adhesion, migration, differentiation, and proliferation (Engler et al. 2006; Ulrich et al. 2009). Conversely, dysregulation of the tissue mechanical environment has been linked to the development of tissue dysplasia and fibrosis, processes that contribute to pathological progression in cancer and cardiovascular diseases (Derrick and Noel 2021; Levental et al. 2009; Paszek et al. 2005). Thus, a deeper understanding of physiological mechanotransduction is critical to understanding disease progression and identifying potential therapeutic targets.

At the cellular level, mechanotransduction begins at the cell surface, where integrins and cadherins couple the ECM and neighboring cells to the cytoskeleton and nuclear envelope (Weber et al. 2011) (Figure 1). Mechanosensitive receptors including ion channels such as Piezo and TRPV, and GPCRs undergo conformational changes under force, activating downstream signaling cascades (Coste et al. 2010; Xu et al. 2018). Forces are relayed via the cytoskeleton, through the LINC complex to the lamina and chromatin, altering nuclear mechanics (Carley et al. 2021; Haase et al. 2016). Cytoskeletal mechanosensing also drives transcriptional co‐activators such as YAP/TAZ to enter the nucleus and reprogram gene expression (Dupont et al. 2011). Together, these pathways illustrate how cells convert external forces into nuclear responses.

FIGURE 1.

Cellular structures involved in mechanotransduction. Simplified view of an epithelial cell showing multiple adhesion complexes that mediate the transmission of forces (tensile, compressive, and shear stresses) between the cell and the surrounding ECM or neighboring cells. Interaction with the ECM is regulated by focal adhesion complexes based on the transmembrane protein integrin. At the cell–cell interface, adherens junctions are regulated by cadherin‐based complexes. Mechanosensitive channels such as Piezo and TRPV contribute to the sensing of tension, curvature, and shear stress. At the nuclear level, mechanical load increases tension transmitted across the LINC complex (linker of nucleoskeleton and cytoskeleton), leading to mechanotransduction at the nuclear pores, nuclear lamina, chromatin, and nucleoli.

Changes in nuclear structure and mechanics inevitably affect subnuclear domains, including the nucleolus, the most prominent nuclear condensate. Nucleolus is the site of transcription and processing of 5.8S, 18S, and 28S ribosomal RNAs (rRNAs), which constitute the most abundant RNA species within the cell. High rates of rRNA transcriptional and processing activities contribute to nucleolar assembly as phase‐separated biomolecular condensates, within which pre‐ribosomal subunits are assembled (Quinodoz et al. 2025; Yao et al. 2019). This organelle maintains complex structural organisation while maintaining constant molecular flux with the surrounding nucleoplasm, allowing rapid transcriptional repression and response to extracellular stressors (Boulon et al. 2010; Lafontaine et al. 2020; Li et al. 2023). Due to its membraneless nature, however, the nucleolus largely bypasses classical mechanotransduction pathways which rely on membrane‐associated mechanosensing.

Although the influence of mechanics on the nucleus is well established, whether such paradigms extend to membraneless condensates like the nucleolus remains unclear. Beyond lamina and chromatin coupling of cytoskeleton, early bead‐displacement assays demonstrated that cytoskeletal prestress and actin organization can directly influence nucleolar mobility, revealing mechanical coupling between the cytoskeleton and nucleolus (Hu et al. 2005; 2004). By contrast, pharmacological perturbation of actin showed minimal changes in nucleolar morphology (Caragine et al. 2019), underscoring that this connection may be context‐dependent. These findings raise important questions regarding both the mechanisms by which external forces propagate to membraneless assemblies like the nucleolus, and the downstream consequences of such mechanical coupling. In this review, we therefore highlight the emerging and underexplored dimension of nucleolar mechanobiology in the context of the cellular mechanical environment. We examine seminal studies that have uncovered mechanically induced regulation of nucleolar structure and function. We highlight the role of ECM cues, cell migration, confinement, and extrinsic mechanical stress on nucleolar structure and function, both ribosomal biogenesis and cell signaling. Given the novelty of this concept, we also highlight evidence from osmotic shock studies, which effectively alter nuclear mechanics and carry strong implications for the nucleolus. Notably, since osmotic shock also mechanically perturbs the nucleus, it may provide inspiration for uncovering the currently unknown molecular pathways underlying nucleolar mechanoadaptation.

2. Nucleolar Structure and Ribosome Biogenesis

The mammalian nucleolus exhibits a canonical tripartite organization comprising the fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC), as established by high‐resolution imaging studies (Thiry and Lafontaine 2005). More recently, in vitro reconstitution studies and live cell imaging have demonstrated that this layered architecture arises from multiphase demixing of biological macromolecules (Feric et al. 2016; Lafontaine et al. 2020; Tchelidze et al. 2017) (Figure 2A). The FC‐DFC sub‐compartments are structurally linked rDNA transcriptional centers which appear to be interspersed within the GC matrix. These distinct nucleolar sub‐compartments (FC, DFC, and GC) serve as sites for the successive stages of rRNA transcription, processing, and ribosome subunit assembly (Thiry and Lafontaine 2005) (Figure 2B). Recent work suggests that the progression of pre‐rRNA processing itself contributes to the emergence and maintenance of nucleolar sub‐compartments (Lafontaine 2019; Quinodoz et al. 2025). Thus, despite lacking a surrounding membrane, the nucleolus exhibits a highly ordered internal organization.

FIGURE 2.

Nucleolar structure and its role in ribosome biogenesis. (A) Schematic of a eukaryotic cell showing the subnuclear membraneless organelle—the nucleolus. Nucleolar internal organization comprises three major compartments: the fibrillar center (FC; green), the dense fibrillar component (DFC; blue), and the granular component (GC; violet). (B) Each of the compartments has a well‐defined function, which is described according to the compartment‐related color code. (C) Directional flux and processing stages impact the rheology of the nucleolus, with entangled pre‐rRNA in the core (small gyration radius; Rg) rendering the nucleolus elastic, while progressive processing stages make the nucleolus more viscoelastic, culminating in the assembly of cleaved pre‐rRNA with ribosomal proteins in the GC, which is characterized by viscous‐like rheology (Lafontaine et al. 2020; Riback et al. 2023).

The nucleolus maintains sufficient physical integrity to persist as discrete structures during biochemical extraction from tissue lysates (Andersen et al. 2002). In contrast, a large body of evidence demonstrates that the assembly of the nucleolus is driven by liquid–liquid phase separation (LLPS) following the nucleation of RNA polymerase I (Pol I) complex at the nucleolar organizer regions (NORs) of acrocentric chromosomes (Lafontaine et al. 2020). The local enrichment of rDNA transcription and ATP‐dependent processes promote high‐valency protein‐protein and protein‐RNA interactions, driving the multiphase separation of subnucleolar compartments (Caragine et al. 2019; Falahati and Wieschaus 2017). This molecular organization is mediated by proteins with intrinsically disordered regions (IDRs), such as fibrillarin (GAR domains) and nucleophosmin (domains containing acidic and basic tracts), which form multivalent RNA–protein interaction networks (Ferrolino et al. 2018; Mitrea et al. 2016; Yao et al. 2019). In vitro, fibrillarin and NPM1 co‐assemble into core‐shell droplets driven by differences in their interaction properties and interfacial tensions, a process that is strongly modulated by RNA and consistent with their distribution within the nucleolus in vivo (Feric et al. 2016). These findings support a model in which active rDNA transcription and ATP‐dependent processes nucleate and facilitate LLPS events at nucleolar organizer regions (NORs).

Beyond this internal phase behavior, nucleolar structure is influenced by structural components of the nucleus, including perinucleolar chromatin and nuclear lamina (Bizhanova and Kaufman, 2021). Perinucleolar chromatin forms a heterochromatin rim, enriched in nucleolus‐associated domains (NADs) which partially overlap with lamina‐associated domains (LADs) (Bersaglieri and Santoro 2019; Vertii et al. 2019), creating a complex chromatin architecture interfacing the nucleolar periphery and the nuclear lamina (Gupta et al. 2025). Disruption of lamin A/C or lamin B perturbs nucleolar architecture, resulting in nucleolar shape abnormalities, fragmentation, and altered rRNA synthesis (Buchwalter and Hetzer 2017; Sen Gupta and Sengupta 2017; Malhas et al. 2007). Thus, nuclear lamina and higher‐order chromatin architecture contribute as critical regulators of nucleolar structure, while also influencing its spatial positioning and functioning.

Importantly, chromatin reorganization accompanies altered nucleolar functioning under stress. For instance, RNA Pol I inhibition pharmacologically with CX‐5461, or with heat shock stress results in remodeling of perinucleolar heterochromatin and redistribution of chromatin modifiers such as polycomb repressive complexes (PRC) (Azkanaz et al. 2019; Potapova et al. 2023; Snyers et al. 2022). Moreover, specific pathways of epigenetic regulation of rDNA transcription have been outlined under stress conditions, further linking nucleolar remodeling to chromatin organization (Boulon et al. 2010; Rolicka et al. 2020; Velichko et al. 2019). Such studies emphasize a strong functional link between chromatin state and nucleolar activity. Because chromatin and lamina are major load‐bearing components of the nucleus which undergo rapid remodeling under mechanistic perturbations, their coupling to the nucleolus provides a plausible route by which mechanical forces reshape nucleolar structure and function.

3. A Biophysical Look at the Stress Responsive Nucleolus

Current literature characterizes the nucleolus as a viscoelastic, phase‐separated biomolecular condensate whose architecture is shaped by active rDNA transcription and multivalent protein–RNA interactions (Lafontaine et al. 2020). Building on this framework, a recent study used micropipette aspiration to further resolve the differential interfacial tension and rheological properties of its sub‐compartments (Cheng et al. 2025). The DFC exhibits viscoelastic solid‐like dynamics, whereas the granular component (GC) behaves like a Newtonian fluid. These rheological differences likely arise from distinct protein distributions and RNA dynamics within each compartment. For example, the hallmark DFC protein fibrillarin displays gel‐like elastic properties in repeated reconstitution assays, implicating it in the DFC's relative rigidity and elastic response (Feric et al. 2016). Additionally, evidence suggests that transcription and processing of rRNA may contribute to these differential nucleolar material properties. Nascent rRNA transcripts form an entangled mesh that becomes progressively less cross‐linked as processing advances, thereby contributing to the compartment‐specific rheology (Riback et al. 2023). Such observations indicate that nucleolar mechanical state is not static, but is dynamically influenced by ongoing active molecular processes that effectively contribute to material properties. Optogenetically induced nucleolar gelation leads to accumulation of unprocessed rRNA transcripts, indicating a tunable, reciprocal interplay between condensate material properties and rRNA maturation (Zhu et al. 2019). Together, these findings underscore the existence of bidirectional feedback between nucleolar function and material state.

This biophysical model gains further depth when the nucleolus is challenged by stress, evidenced by the various characteristic morphological states described during nucleolar stress. A classic example is the formation of nucleolar caps upon Actinomycin D treatment, in which DNA transcription is inhibited and the components of DFC relocalize to the nucleolar periphery (Shav‐Tal et al. 2005). Inhibition of rRNA processing, by contrast, results in a nucleolar necklace morphology, a beaded arrangement of rRNA processing factors around the nucleolar rim (Louvet et al. 2005). Additionally, RNA polymerase II inhibition causes unraveling of fibrillar centers and fragmentation of the granular component (Haaf and Ward 1996). Disruption of proteostasis through proteasome inhibition or protein misfolding triggers the formation of nucleolar aggresomes and amyloid bodies, respectively (Audas et al. 2016; Latonen et al. 2010). These structures are characterized by the selective exclusion of most nucleolar proteins, sequestration of specific proteins and RNAs, and dramatic alterations in intra‐nucleolar phase dynamics (Gavrilova et al. 2024). Together, these stress morphologies illustrate the remarkable plasticity of the nucleolus, undergoing significant reorganization under biochemical perturbations.

Across diverse stresses — heat shock, osmotic changes, DNA damage, or nutrient deprivation — nucleolar structural remodeling is accompanied by redistribution of key proteins such as NPM1 (Yang et al. 2016). To highlight these phenotypes in the context of various cellular stressors, we surveyed publicly available human proteomic databases (UniProtKB and PDB) to identify the reported abundance of proteins within the nucleolar proteome, and summarized a subset of the seven most abundant proteins for which evidence of stress‐associated redistribution has been reported in literature (Table 1). Such nucleolar remodeling events activate well‐characterized stress pathways that fall into two classes: (i) p53‐dependent, where proteins like RPL11, NPM1, or ARF stabilize p53 to regulate cell cycle arrest, senescence, or apoptosis; and (ii) p53‐independent, involving downstream responses by transcription factors such as E2F‐1, MYC, or NF‐κB (Boulon et al. 2010; Yang et al. 2018). These canonical pathways establish the nucleolus as a stress sensor and signaling hub. However, whether mechanical inputs engage similar signaling routes or trigger distinct nucleolar responses remains largely unexplored.

TABLE 1.

Major nucleolar proteins and their role in stress response.

Protein	Functions	Sites of interaction	Protein mobility and type of stress	p53 dep.	Mechanism	Ref
NPM1 (B23)	Ribosome biogenesis; histone chaperone; nucleo‐cytoplasmic transport	N‐terminal oligomerization domain; central acidic IDR; NLS; C‐terminal nucleic‐acid–binding domain	Translocates Out: oxidative stress, DNA damage, serum starvation, heat shock, osmotic stress	Yes	p53 stabilization; DNA repair; apoptosis regulation	Yang et al. 2016
HSPA8 (Hsc70)	Protein quality control; refolding; ubiquitination; protein translocation	N‐terminal nucleotide‐binding ATPase domain; central substrate‐binding domain; C‐terminal helical lid domain	Translocates In: heat shock, oxidative stress	No	ROS protection, Proteostasis	Wang et al. 2018
Nucleolin (C23)	rRNA transcription and maturation; nucleo‐cytoplasmic transport; chromatin remodeling; cell cycle regulation	Basic N‐terminal chromatin‐binding domain; central RNA‐binding domains; C‐terminal GAR/RGG IDR	Translocates Out: heat shock, DNA damage, osmotic stress	Yes and No	p53‐dependent replication inhibition; DNA damage repair; altered gene regulation	Jin et al. 2023; Yang et al. 2008
RPL5/RPL11	Ribosome biogenesis; 60S subunit components	RPL5: N‐terminal globular domain (5S rRNA binding); C‐terminal extension (RPL11 binding) RPL11: Conserved N‐terminal domain of RNA binding proteins; C‐terminal RNA binding homeodomain helix	Translocates Out: ribotoxic stress	Yes	p53 stabilization; cell cycle arrest	Sloan et al. 2013
APE1	Base excision repair; rRNA quality control; gene regulation	Disordered N‐terminal redox domain; C‐terminal AP endonuclease domain	Translocates Out: oxidative and genotoxic stress	Not given	rRNA oxidation and reduced protein translation. DNA damage response	Lirussi et al. 2012; Vascotto et al. 2009
DDX5 (p68)	ATP‐dependent RNA helicase; Gene‐expression regulation	Two conserved domains (1 and 2) with nucleotide/ATP‐binding sites and helicase activity; variable (IDR) N‐ and C‐terminal sequences	Translocates In: p19ARF knockdown	No	Stimulates 47S transcription; increases ribosome output	Saporita et al. 2011
FBL	pre‐rRNA methylation; processing; pre‐ribosome assembly	N‐terminal GAR domain (IDR); central RNA‐binding domain; C‐terminal α‐helical (methyltransferase) domain	Translocates Out: osmotic stress	Not known	Not Known	Jin et al. 2023
p14ARF	Tumor suppressor	N‐terminal domain with hydrophobic clusters (MDM2‐binding); C‐terminal domain (nucleolar localization)	Translocates Out: heat shock, oxidative stress, Oncogenic stimuli	Yes and No	p53‐ dependent and independent cell cycle arrest and apoptosis	Damalas et al. 2011

Emerging evidence from studies on condensate coarsening and mechanobiology suggests that condensate assembly is influenced by biophysical kinetics and nuclear rheology (Lee 2021; Lee et al. 2022; Zhao et al. 2024). At the cellular scale, nuclear mechanical properties are influenced by extrinsic mechanical cues, which are transmitted to the nucleus, primarily through focal adhesions via an integrated cytoskeletal network of actin filaments, intermediate filaments, and microtubules (Buxboim et al. 2017; Wang et al. 2009) (Figure 1). Because the nucleolus is structurally coupled to the nuclear lamina and heterochromatin domains at the nuclear envelope (Bersaglieri and Santoro 2019b), mechanical signals transmitted to the nucleus, which is approximately 10‐fold stiffer than the surrounding cytoplasm and occupies a substantial fraction of cell volume (Lammerding 2011), may influence nucleolar organization. These considerations highlight the nuclear lamina and chromatin — major contributors to overall cellular mechanics — as potential intermediates linking cell‐scale mechanics to the nucleolus (Dahl et al. 2005; Schreiner et al. 2015; Seelbinder et al. 2024; Thorpe and Lee 2017).

Nuclear lamins form a filamentous elastic network at the inner nuclear membrane, which resist large‐scale nuclear deformation, while the viscoelastic chromatin provides internal load‐bearing resistance to small deformations (Stephens et al. 2017). Perturbation of these components induces distinct alterations in the nucleolar morphology (Sen Gupta and Sengupta 2017; Martin et al. 2009; Stephens et al. 2018). For example, Lamin B1 depletion disrupts the internal organization of the nucleolar compartments (Martin et al. 2009). Similarly, loss of the structural LINC component SUN1 at the nuclear envelope induces nucleolar hypertrophy and reduced rRNA synthesis (Matsumoto et al. 2016). Additionally, mechanical perturbation induces dynamic condensation and decondensation of chromatin state, thereby altering nuclear micro‐rheology and modulating force distribution to subnuclear structures, including the nucleolus (Stephens et al. 2018). In contrast, nuclear mechanics is also influenced by the behavior of nuclear condensates, which preferentially localize in the mechanically compliant euchromatic regions (Zhao et al. 2024). Studies on condensate maturation report that growing condensates can physically restructure and exclude surrounding chromatin, thereby altering local nuclear architecture (Strom et al. 2024; Zhao et al. 2025). Furthermore, literature evidence highlights the role of RNA binding proteins in reorganizing chromatin strands via phase separation dynamics, which can modulate transcription and influence cell fate decisions in processes such as differentiation or stress adaptation (Dehingia et al. 2025). Together, this evidence establishes that nuclear condensates — and the nucleolus in particular — are not merely passive, biophysically separated assemblies. Instead, they actively participate in shaping nuclear mechanics.

4. Direct Evidence of Mechano‐regulation of the Nucleolus

Nucleolar assembly is closely linked to nuclear architecture. Because the nucleus is a large viscoelastic organelle, it often acts as a rate‐limiting structure during deformations such as migration through constricted environments (Davidson et al. 2014; Wolf et al. 2013). Nuclear mechanotransduction pathways, including LINC complex signaling and YAP/TAZ nuclear translocation, coordinate these adaptive responses (Dupont et al. 2011). At the mesoscale, nuclear mechanics reflects the composite nature of the nucleus, consisting of a fluid‐like aqueous nucleoplasm, interwoven with a viscoelastic chromatin network, mechanically coupled to the elastic nuclear lamina (Hertzog and Erdel 2023). Although the nucleolus itself has long been viewed mainly as a site of ribosome biogenesis, recent studies show that extracellular mechanical cues can directly perturb its structure and function. Here, we highlight seminal findings demonstrating how ECM‐derived signals, such as surface geometry, stiffness, and topology, as well as mechanically challenging cellular processes such as migration, confinement, and mechanical stress, affect nucleolar structure, composition, and function.

5. Extracellular Matrix Cues

ECM cues regulate cell architecture, ultimately affecting cell size, cytoskeletal rigidity, and nuclear architecture. Pundel et al. observed that these changes directly impact membraneless nucleolar assemblies within the cell (Pundel et al. 2022). Restricting cell adhesion to collagen islands of 20 µm diameter micropatterns compared to 50 µm islands results in reduced nuclear volume and nucleolar coalescence into single enlarged structures (Figure 3; substrate geometry). These changes were associated with decreased ribosomal regulatory gene expression, reduced rDNA transcription, and overall reduction in protein synthesis. Nucleoskeletal components underwent significant reorganization under adhesion restriction, with lamin A/C redistributing to the nuclear periphery and enlarged peripheral H3K9me3 heterochromatin domains, consistent with enhanced chromatin compaction. Particle Image Velocimetry revealed that reduced adhesion and nuclear size scaling were accompanied by increased rigidity of the viscoelastic chromatin network. Conversely, pharmacologically‐induced chromatin decondensation restored nucleolar number and ribosome biogenesis on restrictive micropatterns, indicating that these mechanically induced changes in nucleolar organization are reversible. Similarly, siRNA‐mediated knockdown of the nuclear envelope LINC component SYNE2 (nesprin‐2), decreased nuclear volume and reduced nucleolar number and function irrespective of micropattern size. This indicates ECM‐modulated cytoskeletal architecture influences nucleolar organization through nucleo‐cytoskeletal coupling. Comparable effects were observed upon restricting cell adhesion capacity on softer matrices, where nuclei exhibited smaller volumes and fewer nucleoli. Together, these findings support a model in which ECM‐regulated modulation of nuclear volume contributes to the control of nucleolar number and functionality.

FIGURE 3.

Extracellular matrix cues alter nucleolar assembly and function. (A) Cells grown on constricted micropatterns show altered adhesion and morphology of the nucleus. (B) Confocal immunofluorescence images of HaCaT keratinocytes grown on micropatterned surfaces for 24 h, stained for nucleus (DAPI, blue) and nucleolin (green). Scale bars = 10 µm. (C) Nucleolar fusion events in cells transfected with Nucleolin‐GFP seeded on 20 µm islands. (D) Correlation and R2 (Pearson's coefficient) for nuclear area and nucleoli number per cell combined from HKs cultured on micropatterns, PA gels, or with low or high calcium. (E) Schematic describing spatial and mechanical cues for epithelial monolayers as leader–follower position and matrix stiffness, posing the question of whether mechanosensitive nucleoli regulate protein synthesis. (F) Merged images of nucleolin (color scale representing pixel intensity) and F‐actin (grayscale) in MCF10A cells after 72 h of culture on soft and stiff polyacrylamide gels (scale bar = 20 µm). Insets in these images show 3D Imaris renderings of representative nuclei (transparent), with nucleoli depicted in white (scale bar = 3 µm). (G) Percentage of cells with total nucleolar area ≥30 µm² for each condition after 72 h of culture. (H) Top and side views illustrate a cell cultured on an array of nanopillars with radii ranging from 400 nm to 150 nm. Smaller nanopillars induce higher local curvature of nuclear envelope (NE) beneath the nucleoli, which correlates with increased rRNA synthesis. Large nanopillars produce lower NE curvature which results in reduced rRNA production. Figure A, B, C and D reproduced and adapted from Pundel, et al. (Pundel et al. 2022). Figure E and F reproduced and adapted from Jaecker et al. (Florence Flick Jaecker et al. 2022).

At the population level, Jaecker et al. (Figure 3; substrate stiffness) showed that ECM stiffness and spatial organization similarly regulate nucleolar architecture (Jaecker et al. 2022). In cultured sheets of epithelial cells, the matrix‐adherent “leader” cell population exhibits significantly larger nucleolar areas and higher nucleolar counts per cell than “follower” cells during 72 h of culture. Both populations displayed further enhancement of this nucleolar phenotype on stiffer substrates at 72 h, implicating ECM rigidity as a key driver of nucleolar response, potentially through its effects on cell mechanics and focal adhesion assembly. Moreover, leader cells displayed thicker actin and vimentin networks, underscoring coordinated cytoskeletal remodeling accompanying nucleolar responses. Transmission electron microscopy revealed an elevated number of fibrillar centers in both leader population and on stiff matrices, consistent with heightened nucleolar activity. This was accompanied by increased chromatin compaction and H3K27me3 levels, closely linking mechanotransductive chromatin dynamics and nucleolar remodeling. Notably, Ki‐67 proliferation markers are increased in leader cells and increased across both cell types on stiff matrices, while pharmacological disruption of cell‐cycle progression or chromatin compaction abolished ECM‐driven nucleolar phenotypes. These reports highlight a complex interplay between ECM‐regulated cell‐cycle progression and chromatin compaction underlying nucleolar organization and functioning. In contrast, premalignant MCF10A and malignant cell lines maintained elevated nucleolar size and number regardless of ECM stiffness, recapitulating cancer‐associated nucleolar hypertrophy. Thus, ECM cues regulate nucleolar organization by modulating nuclear volume and chromatin compaction at the single‐cell level (Pundel et al. 2022), and by coordinating cytoskeletal remodeling and cell‐cycle progression in collectives (Jaecker et al. 2022). Together, these findings establish adhesive and stiffness‐dependent control of nucleolar function.

Beyond changes in nuclear volume and nucleolar architecture, intrinsic nuclear topology also contributes to nucleolar structure and ribosome biogenesis. Recent studies demonstrate nuclear envelope invaginations modulate nucleolar function and ribosome biogenesis. For example, Zhuang et al. in their preprint manuscript documented that nucleoli in epithelial cells maintain frequent contact with the nuclear envelope, and categorized these morphologies as flat, dent, and tunnel interactions (Zhuang et al. 2024). Characteristic high‐curvature tunnel invaginations maintain stable nucleolar contact throughout interphase, and exhibit a positive correlation with rDNA transcription levels.

To probe the role of these invaginations on nucleolar activity, Zhuang et al. performed experiments on cells cultured on nanopillar arrays of different radii to generate nuclear deformations of different curvatures (Zhuang et al. 2024) (Figure 3; substrate topology). They reported that high curvature pillars (150 nm radius) generate nuclear tunnel contact with the nucleoli, accompanied by a significant increase in ribosomal biogenesis. Moreover, mechanically induced tunnel invaginations selectively enhanced nucleolar activity within nucleoli in direct contact with the tunnels, while non‐contacted nucleoli within the same nucleus exhibited reduced transcriptional activity. Conversely, breast cancer and progeria cells showed diminished rRNA synthesis on low curvature nanopillars, confirming that substrate curvature directly modulates nucleolar function. Expansion microscopy revealed tunnel‐associated nucleoli were enriched with reorganized lamin B1, nuclear pore complexes and Exportin‐1, facilitating the nuclear invagination phenotype and ribosomal subunit export, respectively. Moreover, these regions further exhibited reduced surrounding heterochromatin, including decreased nucleolus‐associated domains (NADs) and lamina‐associated domains (LADs), indicating an open chromatin architecture that facilitates ribosomal biogenesis. These findings establish that substrate topology can modulate nucleolar function through stable nuclear envelope invaginations, which create spatially enriched environments to support ribosomal transport and assembly, thereby selectively enhancing nucleolar functionality at the resolution of single nucleolus.

Together, these studies establish the ECM as a key regulator of nucleolar structure and activity, acting through nuclear volume control, chromatin remodeling, and cytoskeletal coupling. By linking single‐cell adhesion, substrate stiffness, and collective behavior to nucleolar organization, they highlight how cell and tissue mechanics can directly tune nucleolar assembly and ribosome biogenesis.

6. Cell Migration and Confinement

Migrating cells frequently encounter constricted pores or dense interstitial spaces that impose substantial nuclear deformation. In vitro models of confined migration reveal that nucleoli undergo dynamic reorganization under these conditions (Zhao et al. 2024) (Figure 4A). Severe nuclear deformation, observed during confined cell migration through micropores, induces frequent nucleolar fission and fusion events, consistent with the LLPS model of nucleolar organization (Figure 4B). Notably, fusion events persist after the nucleus exits confinement, whereas nucleoli undergoing fission were observed to subsequently coalesce following nuclear passage through the constriction. The nucleus of migrating cells displays a polarized chromatin architecture with a leading end containing relatively homogenous chromatin distribution, and a trailing end with heterotypic chromatin organization marked by H2B‐mGFP. This spatial chromatin heterogeneity not only creates a variable mechanical landscape, but also promotes spontaneous condensation of phase‐separating proteins in the softer euchromatic regions, irrespective of functional relevance. Optogenetic manipulations using blue light‐induced synthetic Corelet nuclear condensates confirmed that such chromatin landscapes enhance condensate nucleation in migrating nuclei, implicating chromatin distribution as a regulator of nuclear condensate dynamics (Zhao et al. 2024). Moreover, nucleolar condensates tend to lag toward the trailing edge, alongside an increase in perinucleolar heterochromatin accumulation. This localization bias suggests a complex mechanically induced interface between viscoelastic chromatin and nucleolar dynamics during extreme mechanical deformation.

FIGURE 4.

Cell migration and nuclear confinement alter nucleolar dynamics and function. (A) Illustration of nucleoli in the nucleus undergoing mechanical deformation throughout the confined‐migration event. (B) Representative examples of NPM1‐mCherry labeled nucleoli fusing during confined migration. Arrows: fusion events. Scale bar: 5 µm. (C) Illustration of isolated germinal vesicles in oil confined to ∼50% height under a coverslip. (D) Timelapse images of bright‐field and fluorescence microscopy showing regulated nucleolar fusion events. (E) Illustration of transwell‐based protrusion formation and the mechanism of LARP6 mediated translational hotspots. (F) LARP6 co‐localizes with RP‐mRNAs in protrusions. Representative RNA‐FISH and IF co‐staining images of RPL34 mRNA (green) and LARP6 (red) in protrusions and cell bodies of MDA‐MB231 cells. Cell boundaries (dashed lines) were defined from co‐staining with anti‐tubulin antibody. Figure A and B reproduced and adapted from Zhao et al. (Zhao et al. 2024) Figure C and D reproduced and adapted from Brangwynne et al. (Brangwynne et al. 2011). Figure E and F reproduced and adapted from Dermit et al. (Dermit et al. 2020).

Nucleoskeletal F‐actin components significantly contribute to nucleolar organization, as demonstrated by Brangwynne et al. in isolated Xenopus laevis germinal vesicles (Brangwynne et al. 2011). The filamentous network of nuclear F‐actin physically anchors the nucleoli, while immunodepletion or pharmacological inhibition of actin‐polymerization causes multiple nucleoli to coalesce into a single central body. Furthermore, mechanical confinement induced contact between nucleoli is followed by reorganization and fusion of the GCs, producing enlarged nucleoli, while DFCs remain effectively segregated and display additive behavior (Figure 4C). Interestingly, nucleolar coalescence is often delayed, with repeated transient contacts between two nucleoli preceding fusion. This behavior is consistent with the presence of entropic or kinetic barriers to fusion (Figure 4D). In mammalian cells, chromatin is a major nucleoplasmic scaffold that substantially influences nucleolar dynamics. Experimental and theoretical studies demonstrate that the viscoelastic chromatin network promotes nucleation of phase‐separated condensates, while impeding their coalescence by imposing a kinetic barrier to chromatin rearrangement (Arsenadze et al. 2024; Banerjee et al. 2024; Caragine et al. 2018; Shin et al. 2018; Xia et al. 2025; Zhang et al. 2021). Optogenetic experiments further reveal that in inducible condensate systems, droplet growth actively excludes surrounding chromatin, highlighting a mechanical interface between droplets and chromatin that stabilizes the organization of nuclear condensates (Shin et al. 2018). In the context of the nucleolus, such mechanical interface coexists with chromatin inclusion at nucleolar organizer regions. Moreover, recent works characterize a perinucleolar shell compartment, enriched in heterochromatin‐associated proteins at the nucleolar periphery confirming a strong interface between the chromatin and the nucleoli (Stenström et al. 2020). Collectively, these data indicate that the surrounding chromatin provides a complex mechanistic interface influencing nucleolar assembly.

During migration, localized translation of cytoskeletal mRNAs at the leading edge supports protrusion formation and motility (Condeelis and Singer 2005). A similar mechanism was reported by Dermit et al. who demonstrated that in mesenchymal‐like migrating cells on microporous substrates (Figure 4E), the RNA‐binding protein LARP6 directs ribosomal protein (RP) mRNAs to cytoplasmic protrusions, creating hotspots of RP synthesis that feed ribosome assembly in the nucleus (Dermit et al. 2020) (Figure 4F). The newly synthesized ribosomal proteins are subsequently transported to the nucleus for ribosomal assembly. This mechanism links EMT‐associated migration to increased ribosome biogenesis and nucleolar hypertrophy, a phenotype often observed in metastatic cancer.

Together, studies on cell migration and confinement reveal that mechanical deformation of the nucleus reshapes nucleolar architecture through fusion, fission, and chromatin remodeling. In parallel, localized translation of ribosomal proteins during migration couples cytoskeletal dynamics to nucleolar output. These findings position the nucleolus as a central integrator of mechanical stress and biosynthetic demand, with implications for both normal development and cancer invasion.

7. Mechanical Stress

Beyond ribosome biogenesis, the nucleolus acts as a mechanosensitive hub, integrating physical stress signals with cell fate decisions (Boulon et al. 2010). Although limited studies directly probe mechanotransduction at the nucleolar level, some evidence exists that the nucleolus plays a crucial downstream signaling role in regulating cell survival and fate. During cyclic mechanical stress (CMS), the autophagy marker LC3 undergoes translocation into nuclear and nucleolar compartments, accompanied by cellular autophagy activation (Shim et al. 2020). Another mechanotransduction pathway based on focal adhesion kinase (FAK) re‐localization to the nucleolus is activated in breast carcinoma cells (Tancioni et al. 2015). FAK inhibition results in depletion of the nucleolar protein nucleostemin (NS), while other nucleolar proteins such as B23/NPM and nucleolin remain unchanged. In the nucleolus, FAK forms a signaling complex comprising mTOR, Akt, NS, and FAK, suggesting a nucleolar role in coordinating tumor progression and metastasis. Overall, these works illustrate that nucleolus functions as a selective signaling hub for targets beyond ribosomes in mechanically stressed conditions.

Nucleolar function is regulated at several levels, including rDNA transcription, processing, assembly, and ribosomal protein availability. Transcriptomic and qPCR analyses of statically compressed epithelial monolayers reveal significant downregulation of focal adhesion‐ and ECM‐related transcripts, with concurrent upregulation of proteins involved in ribosome biogenesis (Blonski et al. 2021). This demonstrates that cellular mechanical state impacts ribosome biogenesis and nucleolar functionality. In addition to their roles in ribosome biogenesis within the nucleolus, ribosomal proteins also exhibit extra‐nucleolar signaling functions. Zhu et al. report a significant decrease in the levels of ribosomal protein L35 (RPL35; a component of the 60S large ribosomal subunit) in chondrocytes under cyclic mechanical stress, while its overexpression protects the cells against mechanically‐induced senescence and osteoarthritis (OA) development (Zhu et al. 2024). Conversely, siRNA‐mediated knockdown of RPL35 accelerates OA progression, highlighting a significant role of nucleolar proteins in downstream cell fate signaling during mechanical stress responses. Thus, the nucleolus, while incompletely understood in the context of mechanotransduction, demonstrates established links to mechanically‐induced signaling during stress response.

8. Physiological and Pathological Contexts of Nucleolar Mechanobiology

Beyond experimental perturbations, nucleolar mechanobiology also manifests in physiological contexts such as development and tissue growth, as well as in pathological states like cancer, where altered mechanics reshape nucleolar structure and function. Interestingly, a study by Al Jord et al. demonstrated that cytoplasmic forces generated by F‐actin and myosin motors are essential for nucleolar reorganization during mouse oocyte growth and development (Al Jord et al. 2022). Similarly, skeletal muscle hypertrophy drives coordinated growth of the nucleolus along with cell size in response to increased protein synthesis demands (Nakada et al. 2016). These examples suggest that actomyosin activity and cellular growth programs can directly tune nucleolar condensate size and organization.

In pathological contexts, many cancers exhibit nucleolar hypertrophy and elevated ribosome biogenesis to support rapid proliferation (Montanaro et al. 2008). This phenotype may arise not only from oncogenic transcriptional programs but also from aberrant mechanical cues in the tumor microenvironment, which are known biophysical drivers of cancer progression. For instance, treatment of triple‐negative breast cancer cells with lovastatin induces actin depolymerization and G‐actin relocalization to the nucleus, accompanied by nucleolar stress, decreased rRNA synthesis and loss of stem‐like properties (Wang et al. 2024). These findings highlight a potential therapeutic strategy of targeting cytoskeletal–nucleolar coupling to reduce ribosome biogenesis and impair cancer cell adaptability.

Together, these examples illustrate physiological mechanoregulation of nucleolar condensate size and dynamics within cells. In pathological contexts, they suggest that aberrant mechanical cues, which are biophysical drivers of cancer pathology, concurrently influence nucleolar state. They strengthen the view that the nucleolus acts as an integrator of both intracellular mechanics and external microenvironmental cues, with implications for development, tissue plasticity, and disease.

9. Lessons From Osmotic Shock

Among non‐classical stress paradigms, osmotic shock is relevant to mechanobiology studies because it directly imposes mechanical load on the cell and nucleus. Changes in external osmolarity alter nuclear pore permeability, nuclear envelope tension, nuclear volume, and thereby protein concentration and dynamics within the nucleus. Osmotic perturbations thus could provide mechanistic and molecular insight into how nucleolar condensates respond to mechanical and biochemical stress.

Hyperosmotic shock in yeast induces rapid nucleolar shrinkage and chromatin compaction (Thelen et al. 2021), while hypoosmotic stress causes decondensation. It has been further shown that hypoosmotic shock promotes stabilization of single‐stranded R‐loop structures at rDNA loci, triggering nucleolar protein treacle‐mediated activation of ATR/ATM signaling pathways, and subsequent transcriptional silencing of rDNA (Velichko et al. 2019). Osmotic stress is also accompanied by nucleolar remodeling, including dissociation of FC/DFC subcompartments and redistribution of key proteins such as nucleolin and fibrillarin to the nucleoplasm (see Table 1) (Jin et al. 2023; Yang et al. 2008). These effects are reversible upon restoration of osmotic balance, highlighting nucleolar resilience but also its sensitivity to mechanical deformation.

Notably, not all nucleolar components disengage during osmotic perturbation. PQBP5/NOL10 remains nucleolar and functions as an intrinsically disordered scaffold that facilitates reassembly of other nucleolar proteins upon stress release (Jin et al. 2023). This selective retention suggests that protein redistribution is not merely a passive consequence of osmotic imbalance, but a regulated response that helps preserve nucleolar integrity. Although specific mechanotransduction pathways linking osmotic shock to nucleolar function are still unknown, the pattern of stress‐induced remodeling indicates that nucleolar reorganization itself may participate in sensing and adapting to mechanically imposed biochemical changes—where the movement, retention, or release of specific proteins encodes information about the mechanical state of the nucleus. How classical osmotic‐stress pathways (Burg and Ferraris 2008; Kim et al. 2000) interface with this nucleolar adaptation remains to be investigated.

10. Challenges and Perspectives

The primary challenge in studying the nucleolus lies in the absence of membrane boundaries, specific receptor complexes, and fixed architecture. This membraneless nature means that the nucleolar structure and composition are inherently dynamic and context‐dependent. The absence of receptor‐mediated dynamics leaves an organelle primarily defined by local protein concentration and distribution. Consequently, nucleolar behavior is simultaneously governed by active ATP‐dependent processes and material properties of the surrounding nucleoplasm (Caragine et al. 2019). This requires a new paradigm of looking at nucleolar mechanotransduction through the framework of intracellular phase transitions, wherein mechanical forces modulate nuclear and chromatin architecture, intra‐nuclear protein composition and nucleolar function.

Progress in nucleolar mechanobiology depends on methodological advances. Current work often infers nucleolar mechanics indirectly from nuclear/nucleolar deformation or chromatin state. The current landscape of theoretical studies outlining nucleolar mechanics is largely dominated by condensate‐based coarsening models, which emphasize coalescence as the primary mechanism of nucleolar coarsening, facilitated by the surrounding chromatin environment (Arsenadze et al. 2024). Studies on nucleolar coalescence kinetics at short‐timescales outline effective low‐surface tension and liquid‐like behavior of nucleolar droplets, suspended in surrounding nucleoplasm (Brangwynne et al. 2011; Caragine et al. 2018). A complementary study highlights the importance of DNA–protein interactions using bead‐spring polymer computational modeling, particularly protein‐mediated chromatin bridging, as a key determinant of nucleolar phase behavior, relaxation dynamics, and viscoelastic properties (Zhang et al. 2025). Another study reports high resistance of nucleoli to mechanical deformation and strong anchoring to surrounding heterochromatin (Seelbinder et al. 2024). However, systematic investigations of nucleolar deformability and force transmission remain limited. This gap underscores the need for theoretical and computational frameworks that explicitly account for nucleolar mechanics within nuclear mechanotransduction and the cellular response to extracellular mechanical stress. In empirical studies, more quantitative biophysical tools are needed to directly measure nucleolar material properties under mechanical perturbation. Live‐cell imaging, optogenetics, and microfabricated environments will be essential to probe nucleolar behavior in physiologically relevant mechanical contexts. In vivo studies remain scarce, and their expansion will be critical to link nuclear mechanics and nucleolar organization during development, tissue remodeling, and disease.

Finally, future perspectives highlight both fundamental and translational opportunities. Mechanically induced nucleolar remodeling may contribute to physiological processes such as oocyte maturation and muscle hypertrophy, but also to pathological states such as cancer, where nucleolar hypertrophy supports uncontrolled proliferation. Targeting nucleolar function via cytoskeletal or nuclear mechanics — as seen in recent work on actin depolymerization and rRNA suppression in breast cancer cells (Caragine et al. 2019; Wang et al. 2024) — may provide novel therapeutic strategies. Additionally, recent advances have identified condensate‐modifying drugs (c‐mods), which may offer a route toward nucleolar targeting in pathological conditions. These compounds modulate condensate physical properties and macromolecular networks by altering scaffold interactions, condensate composition, protein conformation and position, and post‐translational modifications, thereby disrupting disease‐associated condensate function. However, the nucleolus remains a highly complex structure and the mechanisms driving aberrant nucleolar phenotypes in pathology are not yet fully elucidated, especially from mechanobiology's perspective. Recent advances in machine learning and artificial intelligence may further enhance efforts to optimize selective targeting of the nucleolus in disease contexts (Mitrea et al. 2022). Therefore, unraveling how external mechanical cues reshape nucleolar condensates promises new insight into the integration of mechanics and cell biology, with broad implications for health and disease.

Conflicts of Interest

The authors declare no competing interests.

Use of AI Tools

ChatGPT (OpenAI, GPT‐4) was used under author supervision to assist with language refinement and to suggest potential literature during the drafting process. All references were independently verified, selected, and cited by the authors. Chat‐GPT assigned the list of proteins from UniProtKB with the whole organism protein abundance data (PDB) to identify seven most abundant nucleolar proteins that we manually assigned their function in stress response. In the review, no AI‐generated content, data, or unverified citations were used. The authors take full responsibility for the accuracy and integrity of the manuscript.