Actin filaments, also known as microfilaments, are critical components of the cytoskeleton, involved in various cellular processes such as motility, cell shape maintenance, mechanosensation, interactions with the extracellular matrix (ECM), and other cells via cell-cell adhesions.

Actin filaments are dynamic, filamentous structures formed by polymerizing monomeric globular actin (G-actin) into filamentous actin (F-actin). The dynamic reorganization of actin filaments is essential for proper cellular responses to both intracellular and extracellular signals. This reorganization is heavily dependent on ABPs, which regulate all aspects of filament organization. ABPs control filament assembly (profilin, actin Related Protein 2/3 complex (Arp2/3)), polymerization (formin, vasodilator-Stimulated Phosphoprotein (VASP)), depolymerization (actin-depolymerizing factor (ADF)/cofilin, gelsolin), capping (CP, tropomodulin), cross-linking (scruin, fascin), and branching (Arp2/3) (Pollard, 2016).

Actin filaments are not static, they rapidly polymerize on one end (the plus end), while on the other end (the minus end) a slower polymerization process is accompanied by filament disassembly to a monomeric state. This polarity arises from actin’s ATPase activity. G-actin is a weak ATPase, but its polymerization induces a conformational change that enhances ATP hydrolysis significantly (Guan et al., 2003). ATP-bound F-actin gradually converts to ADP-bound F-actin, with subsequent phosphate release (Blanchoin and Pollard, 2002). ADP-bound F-actin has a high affinity for the ADF/cofilin complex, which promotes filament disassembly (Carlier et al., 1997). Newly formed ends of microfilaments are called barbed; they are hotspots for the majority of the biochemical reactions and can either undergo further elongation or be stabilized by capping proteins that limit subunit addition or dissociation.

Stress fibers represent another important actin-based structure, consisting of contractile bundles of F-actin and myosin II, cross-linked by α-actinin (Naumanen et al., 2008; Sjöblom et al., 2008). These fibers are typically connected to focal adhesions (FAs) and play a crucial role in mechanotransduction - the process by which cells convert mechanical stimuli into biological responses (Blanchoin et al., 2014). FAs are macromolecular multiprotein sites where a cell connects with the ECM through binding between clustered transmembrane adhesion molecules (integrins) and specific FA proteins. FAs are essential for cell adhesion, mechanosensation, and translating mechanical forces on actin fibers into motile forces that drive cell migration (Wang et al., 2001; Legerstee and Houtsmuller, 2021).

Another actin structure that affects nucleus shape and cell fate determination is the perinuclear actin cap (not to be confused with the protein cap structure on the actin barbed ends). The perinuclear actin cap is a highly organized network of thick acto-myosin bundles covering the apical surface of the nucleus in adherent cells (Khatau et al., 2009). This structure is unique due to its direct connections with both the cell periphery and the nuclear scaffold via actin-cap associated focal adhesions (ACAFAs) (Kim et al., 2012) and Linker of Nucleoskeleton and Cytoskeleton (LINC) protein supercomplex (Crisp et al., 2006; Sgarzi et al., 2023) respectively. This organization allows actin cap to facilitate the transmission of forces from the extracellular matrix to the nucleus, influencing nuclear shape, chromatin organization, and tension-dependent signaling pathways, such as YAP/TAZ (Yes-associated protein/transcriptional co-activator with PDZ-binding motif) (Dupont et al., 2011). Sgarzi et al. (2023) demonstrated that in cancer cells with aberrant activation of hepatocyte growth factor receptor (HGFR), the actin cap is disrupted leading to nuclear shape irregularities and impaired cell motility. This disruption correlates with the relocation of YAP1, a key mechanotransducer, from the nucleus to the cytoplasm, resulting in its inactivation. The study demonstrates that HGFR ablation restores proper actin cap formation, YAP1 nuclear localization, and directional cell movement, underscoring the importance of the HGFR-YAP1 axis in regulating cytoskeletal organization and cell motility.

For many years, the presence and function of actin within the nucleus were subjects of skepticism. However, accumulating evidence now underscores the critical importance of nuclear actin (Kelpsch and Tootle, 2018; Venit et al., 2018). Multiple studies have demonstrated that actin is essential for transcriptional machinery through its interactions with RNA Polymerase (RNAP) I (Philimonenko et al., 2004), RNAPII (Hofmann et al., 2004; Zhu et al., 2004), and RNAPIII (Hu et al., 2004). Moreover, actin acts as a regulator of gene expression in response to environmental changes. A recent study revealed that nuclear actin, in conjunction with the actin-binding protein complex Wiskott–Aldrich syndrome protein (N-WASP)/Arp2/3, induces a serum-dependent transcriptional program by scaffolding active and long-lasting RNAPII under serum stimuli (Wei et al., 2020).

Over the past decade, more examples of the role of actin in gene regulation have emerged. A significant amount of data comes from studies on mesenchymal stromal/stem cells (MSCs). While MSCs have the potential to differentiate into various lineages, the balance between osteogenic and adipogenic differentiation represents a particularly well-characterized dichotomy. These studies have highlighted how actin dynamics and cytoskeletal organization can influence the decision between these two fates (Figure 1). For example, it was shown that in MSCs the persistent long-term presence of intranuclear actin induces the Runx2-dependent expression of osteogenic genes such as osterix (Osx) and osteocalcin (Ocn), leading to the acquisition of an osteogenic cell phenotype (Sen et al., 2015). The Arp2/3 complex, which facilitates actin filament branching, is crucial in this process; its absence strongly promotes adipogenesis rather than osteogenesis (Sen et al., 2017; Sen et al., 2024). Another key actin-binding protein essential for genome organization is the nuclear-localized formin diaphanous-related formin 3 (mDia2). mDia2 plays a crucial role in maintaining the integrity of the nuclear actin-lamin structure. Loss of mDia2 disrupts the lamin B1 structure at the nuclear envelope, compromising the actin-lamin nucleoskeleton and subsequently triggering Runx2-dependent osteogenic differentiation in MSCs (Sankaran et al., 2020).

Microtubules are the largest and most rigid components of the cytoskeleton. Similar to actin, they play a crucial role in maintaining cell shape and other various cellular processes, including cell motility, cell division, intracellular transport, and cellular signaling.

Structurally, microtubules are cylindrical hollow polymers composed of approximately 13 linear protofilaments (PFs) formed by α- and β-tubulin dimers (Goodson and Jonasson, 2018). Among the various tubulin isoforms identified, γ-tubulin is particularly significant due to its critical role in nucleating new microtubule structures (Sulimenko et al., 2022). γ-tubulin is highly concentrated in the microtubule organizing center (MTOC) - a cellular region where new microtubules are generated. Centrosomal MTOC plays a major role in cell division.

Similar to actin filaments, microtubes are polarized and dynamic structures. Their assembly dynamics closely resemble those of actin filaments, although microtubules utilize GTP hydrolysis rather than ATP (Hyman et al., 1992). GTP-bound tubulin heterodimers are added to the plus end of the growing microtubule, where they subsequently hydrolyze GTP to GDP, facilitating further depolymerization (Goodson and Jonasson, 2018). The assembly of microtubules is initiated at the MTOC, anchoring the minus ends within the MTOC and orienting the plus ends toward the cell periphery.

Cycles of de/polymerization are frequently interrupted by the sudden switch from growing to quick disassembly, followed by a new growth cycle. This behavior is called dynamic instability and it is believed to enable microtubule tips to efficiently explore cellular space, enhancing their ability to locate and interact with specific targets within the cell (Gudimchuk and McIntosh, 2021).

Microtubules play a fundamental role in intracellular transport, facilitating the movement of organelles, vesicles, and macromolecules within the cell. This transport function is mediated primarily through the interaction of microtubules with motor proteins, such as kinesins and dyneins, which convert chemical energy into mechanical work, enabling the directional movement of cargo along the microtubule tracks. Kinesins generally move cargo toward the plus end of microtubules to the cell periphery while dyneins transport cargo toward the minus end to the MTOC (Hirokawa et al., 2009). Microtubule-associated proteins (MAPs) contribute to the regulation of microtubule-based transport. These proteins can stabilize microtubules, regulate their interactions with motor proteins, and coordinate cargo attachment, thus modulating transport efficiency and specificity (Mandelkow and Mandelkow, 2002).

Aside from the orientation of the mitotic spindle, microtubules' role in cell fate also lies in mechanotransduction, which will be discussed later in the article, and regulation of such important developmental signaling pathways as Wnt. It was shown that dynein interacts with β-catenin, a protein that acts as a transcriptional co-activator in Wnt signaling. Disruption of microtubule dynamics results in improper β-catenin localization, reducing its nuclear translocation and subsequent transcriptional activation of Wnt target genes (Ligon et al., 2001). This regulation is essential in stem cell differentiation and tumorigenesis.

Another pathway through which microtubules affect cell differentiation is Hippo. The Hippo pathway is a key regulator of organ growth, cell proliferation and differentiation, embryogenesis, and tissue regeneration/wound healing, operating through the activity of YAP/TAZ (Fu et al., 2022). Microtubules play a significant role in regulating the subcellular localization of YAP/TAZ: when microtubules are destabilized, YAP/TAZ tends to localize in the cytoplasm, where it becomes inactive, preventing transcriptional activation of its target genes, and conversely, stable microtubules promote YAP/TAZ nuclear translocation, where they activate genes associated with cell proliferation, survival, and stem cell maintenance (Dupont et al., 2011).

IFs differ from actin filaments and microtubules in their structural diversity and functional roles. Regardless of their type, IFs are composed of proteins that form filaments with a uniform diameter of approximately 10 nm and exhibit an organized α-helical conformation, which favors the formation of two-stranded coiled coils, contributing to the greater flexibility and mechanical strength of IFs (Herrmann and Aebi, 2016).

Intermediate filaments are classified into six types based on sequence homology (Szeverenyi et al., 2008).

• Type I and II are acidic and basic keratins, respectively. They are predominantly found in epithelial cells and form heteropolymeric filaments essential for the structural integrity of the epidermis and its appendages.

In contrast to the dynamic nature of actin filaments, the lamin A/C nucleoskeleton appears relatively static in fully differentiated cells until deregulated by aging, cancer, or epithelial-to-mesenchymal transition (EMT) (Heo et al., 2016; Sankaran et al., 2020; Pang et al., 2024). However, subtler rearrangements still occur in response to various stimuli, such as mechanotransduction. The nuclear lamina plays a key role in transmitting biomechanical forces to the cell nucleus and chromatin. It is anchored to the cytoskeleton through a nuclear envelope protein complex called LINC (linker of nucleoskeleton and cytoskeleton) (Mellad et al., 2011). The LINC complex is composed of two families of integral membrane proteins: SUN (Sad1p, UNC-84) and conserved C-terminal KASH (Klarsicht/ANC-1/Syne Homology) proteins. SUN proteins are located in the inner nuclear membrane, where they interact with lamins. They extend into the perinuclear space, where they interact with KASH proteins, which are anchored in the outer nuclear membrane. The KASH proteins then extend into the cytoplasm, where they engage with the cytoskeleton (Bouzid et al., 2019; King, 2023). Lamins, especially lamin A, are critical mediators in mechanotransduction; the integrity of the lamina affects how the nucleus and the cell respond to mechanical stress, such as shear stress or substrate stiffness (Donnaloja et al., 2020; Sapra et al., 2020). Indeed, matrix stiffness directly influences lamin A protein levels: stiff substrates increase lamin A levels, leading to osteogenic differentiation of stem cells, whereas soft matrices are associated with low lamin A levels and adipogenic differentiation (Engler et al., 2006; Swift et al., 2013). These findings correlate with results from experiments involving knockdown and overexpression of the lamin A gene, LMAC: LMAC deficiency promotes adipocyte differentiation, while overexpression of LMAC increases osteogenic differentiation (Scaffidi and Misteli, 2008; Akter et al., 2009; Swift et al., 2013; Tsimbouri et al., 2014).

Chromatin structure plays a crucial role in key cellular processes by regulating accessibility to DNA, thereby influencing the interactions of proteins and other factors that are essential for development and differentiation. Chromatin-modifying complexes are responsible for activating and repressing transcription at specific chromosomal regions through epigenetic modifications (Klages-Mundt et al., 2018). These complexes can be categorized into subfamilies based on their central ATPase components. Among them, SWItch/Sucrose Non-Fermentable (SWI/SNF) complexes are particularly significant due to their involvement in various processes, including transcriptional regulation and the modulation of genes associated with cell adhesion and ECM proteins (Alfert et al., 2019). Furthermore, SWI/SNF complexes containing Brahma-related gene 1 (BRG1) and BRAHMA (BRM) ATPase units are critical for regulating the expression of genes necessary for cellular proliferation and differentiation, establishing their close association with cell cycle regulation (Hogan and Varga-Weisz, 2007).

There is substantial evidence indicating a correlation between chromatin-modifying complexes and cytoskeletal proteins, particularly nuclear actin. Β-actin is a ubiquitously expressed isoform of actin found in the nucleus, where it plays several significant roles, particularly in shaping the chromatin landscape (Dugina et al., 2022). Actin and ARPs play crucial roles in the assembly and regulation of chromatin-modifying complexes (Klages-Mundt et al., 2018), facilitating transitions between transcriptionally active chromatin compartments (A-compartments) and repressed chromatin compartments (B-compartments), which correspond to increased and decreased gene expression, respectively. Notably, several key regulators of cell differentiation, such as SRY-Box Transcription Factor 21 (SOX21), Bone morphogenetic protein 3 (BMP-3), and BMP-6, are among the genes influenced by these chromatin landscape changes (Sen et al., 2024). Actin-binding proteins are essential in the recruitment, assembly, and maintenance of the structural integrity of chromatin remodeling complexes (Pollard, 2016). The relationship between ARPs and chromatin-modifying complexes was thoroughly reviewed by (Pollard, 2016). ARPs can form heterodimers with each other, such as ARP7-ARP9, or pair with actin, as in the Actin-ARP4 complex, to promote the structural integrity of chromatin-modifying complexes (Clapier and Cairns, 2009; Schubert et al., 2013). Among these, ARP4 is the most commonly identified ARP in chromatin-modifying complexes, where the Actin-ARP4 pair interacts with the HSA domain unique to each complex (Farrants, 2008).

Actin and ARPs also affect chromatin regulation independently of their physical involvement in chromatin-modifying complexes by directly interacting with histones (Klages-Mundt et al., 2018). The absence of β-actin leads to the downregulation of epigenetic marks for active chromatin such as acetylation on lysine 9 of histone H3 (H3K9ac) and trimethylation of lysine 4 on histone H3 (H3K4me3) at rDNA loci. While epigenetic marks for repressive chromatin such as monomethylation of lysine 4 on histone H3 (H3K4me1) were found to be upregulated (Almuzzaini et al., 2016). Moreover, knockout of β-actin in mouse embryonic fibroblasts leads to alteration of the heterochromatin landscape across the genome compared to wild-type cells, specifically accompanied by increased methylation of histone 3 (H3K9Me3) levels in the majority of chromatin regions (Xie et al., 2018a). This increased H3K9me3 methylation is also implicated in the induction of neural gene programs. Directly reprogrammed β-actin knockout embryonic fibroblasts into neurons contain increased levels of H3K9Me3 along with loss of the ATPase subunit of the chromatin-modifying complex BAF at transcription start sites of multiple gene loci (Xie et al., 2018b). Reduction of cytoskeletal tension using chemical agents like blebbistatin in primary fibroblasts being differentiated into neurons downregulated the expression of heterochromatin genes manifested by the decreased marks H3K27me3 and H3K9me3, while promoting an open chromatin structure globally and locally, manifested by an increase in AcH3, H3K4me3, and H3K4me1 marks. Furthermore, Blebbistatin treatment increased histone acetyltransferase (HAT) and H3K4-specific histone methyltransferase (HMT) activity while reducing histone deacetylase (HDAC) and histone demethylase (HDM) activity. This could lead to increased histone H3 acetylation and H3K4 methylation, promoting gene activation. Additionally, it increased accessibility at the promoter or enhancer regions of neuronal genes (Soto et al., 2023).

Besides actin, Nuclear myosin 1 participates in the recruitment of histone acetyltransferases (HATs) and histone methyltransferases (HMTs) which promote an epigenetic landscape compatible with active transcription (Almuzzaini et al., 2015). NM1 was also found to be a part of chromatin modifying complex B-WICH which interacts with actin forming an actomyosin molecular motor affecting the attachment of RNA polymerase 1 with the chromatin (Ye et al., 2008).

Furthermore, studies have found the cell geometry to affect the nuclear-cytoplasmic relocalization of SET And MYND Domain Containing 3 (SMYD3) lysine methyltransferase in murine myoblasts. This distribution in response to cell geometry was correlated with cytoplasmic and nuclear lysine tri-methylation levels and it could change SMYD3 substrates and subsequent nuclear vs. cytoplasmic functions (Pereira et al., 2020). Cell geometry has also been found to affect cytoplasmic-to-nuclear redistribution of histone deacetylase 3 in an actomyosin-dependent manner which in turn affects chromatin compaction (Jain et al., 2013). Another study demonstrated that applying cyclic stretch to fibroblasts during reprogramming into induced pluripotent stem cells (iPSCs) significantly boosted reprogramming efficiency. The stretched cells showed epigenetic modifications, particularly a reduction in H3K9me3, along with global and gene-specific changes in chromatin occupancy, which contributed to the improved generation of iPSCs (Park et al., 2023).

Chromatin organization and maintenance also rely on lamins: lamin A/C is predominantly associated with euchromatic regions, whereas lamin B is primarily linked to heterochromatin (Gesson et al., 2016). Maintaining the nuclear organization depends on several components, one of which is nuclear lamina and specialized topologically associating domains named lamina-associated domains (LADs) (van Steensel and Belmont, 2017; Rowley and Corces, 2018). This organization is vital for regulating gene expression by controlling the accessibility of DNA to transcription factors and other regulatory proteins.

Although LADs often interact with the nuclear lamina, these two entities are distinct and should not be confused. The nuclear lamina is a network underlying the nuclear envelope, while LADs are heterochromatin regions located at the nuclear periphery, characterized as transcriptionally repressed and enriched with repressive histones such as H3K9me2, H3K9me3, and H3K27me3 (van Steensel and Belmont, 2017). LADs are distributed along heterochromatin on chromosome arms but are not associated with pericentromeric heterochromatin (Guelen et al., 2008; Peric-Hupkes et al., 2010; Ho et al., 2014).

The molecular mechanisms underlying the transcriptional regulation of LAD are not completely discovered yet, but there is strong evidence that at least one of the mechanisms is dependent on dynamic binding to the nuclear lamina, however not limited by it due to weak correlation between nuclear lamina loosening and changes in gene expression that was shown in several recent studies (Forsberg et al., 2019; Chang et al., 2022).

Regulators of lamina binding can be broadly classified into two categories: tethers and looseners (Manzo et al., 2022). Lamins themselves, particularly lamin B and C, play a significant role in anchoring LADs to the lamina, but they are not the only regulators involved (Ulianov et al., 2019; Wong et al., 2021; Chang et al., 2022). Lamin B Receptor (LBR) appears to be one of the most crucial tethers in mammals. Studies have shown that knocking out or down-regulating LBR disrupts the organization of LADs, leading to abnormal chromatin remodeling and gene expression (Solovei et al., 2013; Falk et al., 2019; Herman et al., 2021; Schep et al., 2021). Notably, LBR deficiency is strongly associated with cellular senescence (Arai et al., 2019; En et al., 2020), highlighting the importance of proper chromatin organization for cellular health and suggesting that LBR’s role in suppressing genome instability could make it a potential target for promoting cellular longevity.

Several other proteins have been identified as potential tethers of LADs, including chromo domain-containing protein Ces-4 in Caenorhabditis elegans (Bian et al., 2020) and proline-rich 14 (PRR14) (Poleshko et al., 2013; Dunlevy et al., 2020), PR/SET Domain 16 (Prdm16) (Biferali et al., 2021), and Zinc Finger With KRAB And SCAN Domains 3 (ZKSCAN3) (Hu et al., 2020) in mammals. The exact mechanisms by which these proteins tether LADs remain to be fully elucidated, though evidence suggests that these mechanisms may involve the recognition of H3K9me marks on heterochromatin (Poleshko et al., 2019; Bian et al., 2020; Biferali et al., 2021).

Specific looseners of LADs have yet to be identified, but it has been shown that forced gene activation within a LAD can cause local detachment of chromatin from the nuclear lamina, affecting around 50 kb flanking the activated site, and vice versa (Brueckner et al., 2020; Su et al., 2020). Histone acetylation is another factor that appears to weaken chromatin-nuclear lamina interactions. For example, in C. elegans, the loss of the euchromatin binder Mrg1, which normally sequesters histone acetyltransferases, results in increased histone acetylation and subsequent LAD detachment (Cabianca et al., 2019). Similarly, in mammalian cells, depletion of histone deacetylase SIRT3 increases accessibility in LADs, possibly due to increased histone acetylation (Diao et al., 2021).

In understanding the role of the cytoskeleton in cell fate alteration it is important to observe natural cases of mature cytoskeleton rearrangement. Here we will describe an epithelial-to-mesenchymal transition (EMT) - a process that is based on such significant morphological alterations as malignancy and re-epithelialization during wound healing.

EMT is a biological process during which apical-basal polar epithelial cells undergo multiple transcriptional, biochemical, and morphological changes that enable them to weaken strong cell-to-cell junctions, detach from the basal membrane, and obtain a back–front polar mesenchymal cell phenotype, which enhances cells’ migratory capacity due to their dynamic attachment to the extracellular matrix, invasiveness, elevated resistance to apoptosis, and increased production of ECM components (Kalluri and Weinberg, 2009). EMT occurs normally during early embryonic development as well as during wound healing in adults, cancer pathogenesis, and tissue fibrosis. It is important to note that the transition from an epithelial to a mesenchymal phenotype is often incomplete, resulting in cells that occupy various intermediate states depending on their biological context (Nieto et al., 2016).

Cells undergo EMT in response to environmental factors that activate EMT-inducing signaling pathways. The most prominent and well-known pathways include the transforming growth factor-beta (TGFβ) and Wnt pathways, which trigger the expression of EMT-specific transcription factors (TFs) such as Snail, Slug, and Twist 1/2, among others (Díaz-López et al., 2014).

Although the dynamics of cytoskeletal changes during EMT have been a subject of considerable interest [reviewed in Datta et al. (2021)], some aspects, like cytoskeleton dynamics during EMT or the role of microtubules during EMT remain underexplored. Induced EMT stimulates a massive reorganization of actin into stress fibers and their alignment in both cancerous and non-cancerous cell lines (Nieto et al., 2016). These aligned thick actin fibers help form prominent protrusions at the leading edge, which are essential for cell mobility and migration (Datta et al., 2021).

Changes in FAs during EMT depend on cell type greatly. However, two morphological features are common across studied cell lines: a decrease in the average area of FAs and an increase in the frequency of FAs (Geiger et al., 2008; Bianchi et al., 2010; Nurmagambetova et al., 2023). There is a hypothesis suggesting a positive correlation between the size of FAs and cell speed, but the data remains inconclusive (Kim and Wirtz, 2013; Nurmagambetova et al., 2023).

The most significant changes during EMT occur in the organization of microtubules. The spatial organization of the microtubule array becomes more radial, especially at the cell edges. After EMT, cells display a larger area covered by microtubules, while their density at the cell edges decreases. This could indicate microtubule growth in the cell interior rather than at the periphery (Kiss et al., 2018; Nurmagambetova et al., 2023).

Although cytoskeletal remodeling is generally considered a downstream event in EMT, regulated by specific signaling cascades, several examples of the cytoskeleton directly regulating EMT have been documented. For instance, Kelch Like Family Member 23 (KLHL23), an inhibitor of actin polymerization, inversely suppresses EMT (Peng et al., 2018). Further investigation revealed that actin remodeling promotes EMT through the induction of hypoxia-inducible factor and Notch signaling in a cell-density-dependent manner. Actin remodeling also contributes to the disruption of E-cadherin at cell-cell adhesions, facilitating cell detachment from its microenvironment (Yilmaz and Christofori, 2009).

Pascual-Reguant and colleagues discovered a new type of LADs called euchromatin LADs (eLADs), which are formed by lamin B1 and euchromatin regions (Pascual-Reguant et al., 2018). eLADs are dynamic and change during TGF-β-induced EMT: as EMT begins, the amount of lamin B1 increases at TAD borders, strengthening these borders. Over time, additional eLADs form around transcriptionally active genes involved in the EMT pathway. Once cells acquire a mesenchymal phenotype, there is a tendency for these eLADs to become inactive. These findings suggest that lamin B1 might play a critical role as an architectural protein in establishing new genomic conformations and transcriptional patterns pivotal for new cell types during EMT (Pang et al., 2024).

Given that EMT represents a natural example of transdifferentiation, where cells undergo significant cytoskeletal rearrangements, signaling cascades, and changes in nuclear architecture, these processes offer valuable models for studying cellular plasticity. Leveraging factors and signaling pathways associated with EMT and cytoskeletal remodeling components as additional reprogramming agents can potentially drive transdifferentiation in controlled settings, thus facilitating new protocols for cell fate manipulation in regenerative medicine and tissue engineering.

The mechanical environment surrounding the cell, including the properties of the ECM and external forces such as tension, compression, and shear stress, significantly influence cellular behavior, including differentiation and identity switching (Engler et al., 2006; Melo-Fonseca et al., 2023) (Figure 2). The link between mechanical forces and gene expression is mediated by mechanosensitive proteins and signaling pathways in mechanotransduction, which involves the conversion of mechanical signals into biochemical signals via the cytoskeleton. FA complexes anchor the cytoskeleton to the ECM and play a key role in sensing mechanical cues. These complexes can activate a cascade of intracellular signaling pathways, including the Rho/ROCK and YAP/TAZ pathways (Dupont et al., 2011).

Rho signaling specifically facilitates actin polymerization and the formation of stress fibers, essential for maintaining cell shape and helping regulate the contractility required for cells to sense and respond to matrix stiffness and mechanical forces (Xie et al., 2023). Moreover, Rho/ROCK activation affects YAP localization (Nardone et al., 2017), which functions as a mechanosensor and translocates from the cytoplasm into the nucleus in response to mechanical forces (Elosegui-Artola et al., 2017) (Figure 1). Subsequently, the actin filament network is rearranged and aligned perpendicular to the direction of mechanical stress, as demonstrated in hMSCs under tensile stress (Parandakh et al., 2017).

As a result, mechanical pathways converge in the nucleus, where chromatin stretches and unfolds, influencing the transcriptional apparatus and further activation of gene expression (Tajik et al., 2016). During mechanical stimulation, the link between the nucleus and the cytoskeleton is mediated by the LINC complex (Crisp et al., 2006), which translates these signals into intracellular responses. This sensitivity is particularly pronounced concerning substrate stiffness, where LINC complexes promote mechanosensitive gene expression, suggesting that they are integral to the cellular perception of environmental mechanical cues (Alam et al., 2016).

During development, genetic programs and soluble morphogens regulate proliferation, differentiation, and tissue organization. However, mechanical forces impact numerous cell functions (Wozniak and Chen, 2009). For example, stiffness is important during embryogenesis, Cells sense stiffness through actomyosin-based contractility linked to integrin adhesions, which cluster in response to substrate stiffness, activating mechanosensitive proteins and downstream transcription factors (Janmey et al., 2020). In vitro studies use different kinds of substrates to study the interaction between cells and their environments, however, tissues and ECMs exhibit more complex mechanical behaviors, including viscoelasticity and non-linear elasticity which are critical during development and disease (Chaudhuri et al., 2020). For example, during Xenopus laevis gastrulation, mesoderm and notochord stiffness prevent buckling, while the involuting marginal zone stiffens to maintain structural integrity (Adams et al., 1990). Mechanical feedback also plays a crucial role in regulating proliferation; during Drosophila melanogaster oogenesis, localized myosin-generated tension drives epithelial cell proliferation to accommodate tissue growth, while reduced myosin activity suppresses proliferation and deforms tissues, suggesting tension promotes growth while compression slows it (Wang and Riechmann, 2007). Furthermore, in D. melanogaster gastrulation, endogenous tissue compression during germband extension (GBE) upregulates Twist expression, a key regulator of mesoderm and midgut differentiation (Desprat et al., 2008). Furthermore, the effect of mechanical stimulation can push cells into a malignant state, a recent study demonstrated that in a 3D breast cancer culture model, a stiff ECM induces a tumorigenic phenotype by altering the chromatin state. Increased ECM stiffness leads to cells with more wrinkled nuclei and increased lamina-associated chromatin. Cells grown on stiff matrices exhibited more accessible chromatin regions with Sp1-binding footprints. This transcription factor, in conjunction with histone deacetylases 3 and 8, plays a key role in regulating stiffness-induced tumorigenicity (Stowers et al., 2019). Tumor cells in turn progressively remodel cytoskeletal structures and reduce cellular stiffness during tumor progression, which makes targeting cytoskeletal components a target in controlling tumorigenic potential in vivo. Weakening/strengthening actin cytoskeleton can facilitate β-catenin nuclear/cytoplasmic localization, β-catenin in turn binds to the promoter of Oct4, activating it and sustaining self-renewal and malignancy (Chen et al., 2023). To further understand how the cellular environment affects cellular processes, we review in the following section some of the mechanical forces and their effects on cells in vitro.

In addition to physical factors, various biochemical agents can mimic cytoskeletal remodeling effects induced by biophysical stimuli, affecting cell shape and ultimately regulating lineage commitment (Table 1) (Figure 2). It has been well-established that changes in cell shape impact both proliferation and differentiation, largely through the modulation of Rho family GTPases and ROCK-mediated cytoskeletal tension (Sordella et al., 2003; McBeath et al., 2004). For instance, studies on mice deficient in p190-B RhoGAP, an inactivator of Rho, have shown decreased adipogenesis and increased myogenesis in embryonic fibroblasts (Sordella et al., 2003). Moreover, low ROCK activity, which corresponds with decreased myosin light chain phosphorylation, has been reported to influence MSCs' fate during differentiation (Bhadriraju et al., 2007).

MSCs, serve as a well-studied model for investigating how cytoskeletal changes affect fate determination. For example, the small molecule cytochalasin D, which inhibits F-actin polymerization, has been shown to favor adipogenic differentiation of MSCs. Recent reviews (Pampanella et al., 2024) have thoroughly examined cytochalasin’s effects on stem cell differentiation. Similarly, agents like blebbistatin (a myosin II inhibitor) and Y-27632 (a ROCK inhibitor) have been found to decrease osteogenesis while increasing adipocyte-like phenotypes in cells (Kilian et al., 2010). In contrast, nocodazole, a microtubule depolymerizing agent, enhances cell contractility, favoring osteogenic differentiation (Kilian et al., 2010).

Paclitaxel, another ROCK inhibitor, induces aggregation and branching of actin filaments, driving MSCs toward osteoblast differentiation. On the other hand, actin debranching tends to favor adipogenesis (McBride et al., 2008; Sen et al., 2017). These findings suggest that cell fate decisions are closely tied to actomyosin contractility, with the polymerization state of actin or tubulin serving as indicators of differentiation potential. A clear trend has emerged: increasing actin or microtubule polymerization promotes osteogenesis, whereas inhibiting polymerization supports adipogenesis. However, the decision for MSCs to commit to a specific lineage is far more complex than simply modulating actin or tubulin polymerization through chemical agents (Putra et al., 2023).

The effects of cytoskeletal remodeling vary significantly depending on the cell type. For -example, treating human pluripotent stem cells (hPSCs) with Latrunculin A during β-cell differentiation resulted in higher expression of NEUROG3, a marker of late pancreatic development (Hogrebe et al., 2020). In the same study, hPSCs treated with actin and microtubule polymerization inhibitors produced cells at different stages of differentiation within one population, with proportions influenced by the stages of actin and tubulin depolymerization (Hogrebe et al., 2020). In contrast, applying these agents to primary fibroblasts being reprogrammed into neurons yielded different outcomes. Specifically, blebbistatin and Y-27632 reduced intracellular tension and improved reprogramming efficiency, while nocodazole and cytochalasin D disrupted cell division, increased stress fibers, and ultimately reduced reprogramming efficiency (Soto et al., 2023). Reduction of cytoskeletal tension in primary fibroblasts using blebbistatin downregulates heterochromatin genes, decreases H3K27me3 and H3K9me3 marks, and promotes an open chromatin structure. This is reflected by increases in AcH3, H3K4me3, and H3K4me1 marks, as well as heightened HAT and HMT activity. Blebbistatin also reduces HDAC and HDM activity, ultimately promoting gene activation by enhancing histone acetylation and methylation (Soto et al., 2023). Notably, reducing actin cytoskeletal tension during the early stages of reprogramming facilitated a more open chromatin structure, decreased DNA methylation and heterochromatin marks, and increased euchromatin marks at neuronal gene promoters, enhancing reprogramming efficiency (Soto et al., 2023).

The underlying mechanisms of these effects are multifaceted. FA assembly is highly dependent on the force exerted between cells and their environment (Dumbauld et al., 2010). Inhibition of myosin II with blebbistatin significantly reduces vinculin localization to FA and decreases FA area on stiffer substrates (Zhou et al., 2017). Y-27632 treatment interferes with the recruitment of αTAT1, an acetyltransferase involved in microtubule acetylation, to FA and disrupts its interaction with talin (Seetharaman et al., 2022). Inhibitors of actin polymerization, such as cytochalasin D or latrunculin B, have been shown to activate protein kinase C alpha, promoting chondrogenic differentiation in chick embryo MSCs while favoring adipogenesis over osteogenesis in human bone marrow-derived MSCs (Lim et al., 2000; Sonowal et al., 2013). Both cytochalasin D and Latrunculin A also inhibit ERK and AKT activation, which are critical pathways for controlling cell proliferation, differentiation, and survival (Müller et al., 2013).

Moreover, disrupting the actin cytoskeleton with latrunculin A has been linked to reduced ERK1/2 phosphorylation and the subsequent decrease in cell proliferation in both human and murine rhabdomyosarcoma (Würtemberger et al., 2020). Nocodazole treatment has been shown to increase Ppar-γ expression and enhance BMP-2 promoter activity, leading to increased bone formation through the hedgehog signaling pathway (Zhao et al., 2009).