Visualizing nuclear mechanotransduction with FRET-based biosensors

Abstract

Cells continuously sense and respond to mechanical cues from their microenvironment, converting physical stimuli into biochemical signals that regulate gene expression and cellular behavior. This review focuses on the structural and molecular basis of nuclear mechanotransduction and highlights recent advances in fluorescence resonance energy transfer (FRET)-based biosensors developed to interrogate these processes in living cells. We conceptualize nuclear mechanotransduction as three interconnected pillars: (i) direct force transmission through the LINC complex, (ii) structural mechanosensing mediated by nuclear deformation involving the nuclear lamina and nuclear pore complex, and (iii) transcriptional mechanotransduction driven by epigenetic regulation and mechanosensitive signaling pathways. Because mechanical signals are rapid, spatially localized, and highly dynamic, FRET has emerged as a powerful approach for real-time visualization of nuclear mechanics. Its sensitivity to nanoscale distance and molecular orientation enables detection of force-induced protein rearrangements and dynamic structural transitions within the nucleus. Accordingly, we review recent FRET-based strategies for probing force transmission at the nuclear envelope, lamina strain, and LEM-domain/BAF protein dynamics, as well as biosensors reporting mechanically regulated histone modifications and YAP nuclear localization. Together, this review provides a unified framework for understanding nuclear mechanotransduction and underscores the expanding role of FRET-based tools in dissecting nuclear mechanics with high spatiotemporal resolution.

Introduction

Tissues in the human body experience distinct mechanical and biochemical microenvironments that reflect their specialized physiological functions. ¹ Although cells share an identical genome, they adapt to the physical and chemical properties of their local niches, thereby establishing tissue-specific organization and differentiation programs.^2,3 Cells continuously sense and respond to a wide range of mechanical cues, including extracellular matrix (ECM) stiffness, shear stress, tensile and compressive forces, and substrate deformation. These physical inputs are translated into biochemical signaling events that ultimately regulate gene expression. ⁴ Mechanical cues not only modulate tissue-specific signaling pathways but also critically influence morphogenesis, cell migration, polarization, and proliferation.^5–8 Consequently, elucidating how cells detect and interpret mechanical stimuli is essential for understanding intracellular signaling in physiologically relevant contexts.

Mechanosensing refers to the cellular detection of external mechanical stimuli, whereas mechanotransduction describes the conversion of these physical signals into intracellular biochemical responses. ⁹ When mechanical forces are transmitted to the nucleus and subsequently regulate gene expression, this process is termed nuclear mechanotransduction.^9,10 Nuclear mechanotransduction can be broadly organized into three interconnected aspects: (i) direct mechanical force transmission to the nucleus, (ii) nuclear deformation and structural mechanosensing, and (iii) transcriptional mechanotransduction mediated by mechanosensitive nuclear signaling pathways. Because mechanical signals arise and propagate on rapid timescales and within spatially confined intracellular compartments, investigation of nuclear mechanotransduction requires live-cell approaches with high temporal resolution. ¹¹ Moreover, efficient force transmission depends on the physical continuity of molecular assemblies spanning from the plasma membrane to the nuclear interior.^12,13

Fluorescence resonance energy transfer (FRET) is a distance- and orientation-dependent phenomenon that occurs when the emission spectrum of a donor fluorophore overlaps with the excitation spectrum of an acceptor fluorophore and the two fluorophores are positioned within approximately 10 nm of each other. Under these conditions, excitation of the donor leads to non-radiative energy transfer to the acceptor, producing characteristic changes in fluorescence signals. ¹⁴ Based on this principle, genetically encoded FRET biosensors (FRET-GEBs) have been developed to visualize dynamic signaling processes in living cells.^15,16 Owing to their exceptional sensitivity to protein conformational changes, intermolecular spacing, and dynamic rearrangements within multiprotein complexes, FRET-GEBs are particularly well suited for probing mechanotransduction.^11,17–19 In this review, we summarize current concepts of nuclear mechanotransduction and highlight recent FRET-based studies that have provided mechanistic insights into how mechanical forces are sensed, transmitted, and processed at the nuclear level (Figure 1).

Figure 1.

Schematic overview of fluorescence resonance energy transfer (FRET)-based approaches for investigating nuclear mechanotransduction. Genetically encoded FRET biosensors enable real-time visualization of how mechanical signals propagate from the cytoskeleton to the nucleus. The left panels illustrate representative FRET strategies for detecting molecular tension and interactions at the nuclear envelope, including nesprin stretching within the LINC complex, lamin deformation within the nuclear lamina, and emerin–LAP1/BAF associations. The central panel summarizes the hierarchical framework of nuclear mechanotransduction, depicting transmission of mechanical cues from cytoskeletal structures to nuclear architecture and gene regulatory machinery. The right panels highlight FRET-based probes for monitoring downstream signaling events, such as YAP/TAZ nuclear localization and histone epigenetic modifications. Together, these approaches demonstrate how FRET enables dynamic, spatiotemporally resolved analysis of nuclear mechanotransduction in living cells. Schematic overview was created with BioRender (https://www.biorender.com).

Nuclear mechanotransduction: From force transmission to transcriptional regulation

To establish a comprehensive mechanistic framework for nuclear mechanotransduction, it is essential to delineate how extracellular and intracellular forces are physically conveyed to the nucleus, how these forces deform nuclear structures, and how such deformation ultimately reshapes chromatin organization and transcriptional programs. Rather than acting as a passive repository of genetic material, the nucleus functions as an integrated mechanical organelle that continuously senses, transduces, and responds to physical cues from the cellular microenvironment. This multistep process directly links force transmission at the plasma membrane to biochemical signaling and transcriptional outputs within the genome.

Direct mechanical transduction to the nucleus

Physical forces generated at integrin-mediated focal adhesions are initially propagated through the actin cytoskeleton, which serves as the principal load-bearing network of the cell. Actomyosin contractility generates tensile stresses that are distributed across interconnected cytoskeletal systems, including intermediate filaments and microtubules, thereby forming a mechanically integrated scaffold spanning the entire cytoplasm. Through this architecture, forces arising from extracellular matrix rigidity, cell–cell interactions, or intracellular contractility are transmitted over long distances with minimal mechanical dissipation.^13,20,21

These cytoskeletal forces are subsequently conveyed to the nucleoskeleton via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex embedded within the nuclear envelope.^13,22 The LINC complex establishes a continuous physical bridge spanning both nuclear membranes, directly coupling cytoplasmic filaments to intranuclear structural elements. A key contributor to this transmission is the perinuclear actin cap—a specialized dome-like array of highly contractile apical stress fibers overlying the nucleus. The actin cap forms direct connections with the LINC complex and functions as a dominant force-transmitting structure that constrains nuclear shape, aligns nuclear orientation, and rapidly modulates nuclear morphology in response to changes in cellular tension.^23,24

At the molecular level, spectrin-repeat–containing nuclear envelope proteins known as nesprins anchor actin filaments at the outer nuclear membrane (ONM) and interact with Sad1p and UNC-84 homology (SUN) proteins at the inner nuclear membrane (INM). SUN proteins further associate with lamins and chromatin, thereby forming a continuous mechanical linkage from the cytoskeleton to the nuclear interior. ²⁵ Through this highly organized and mechanically integrated architecture, physical stimuli experienced at the cell surface can be transmitted to the nucleus within milliseconds, enabling rapid nuclear responses that operate independently of slower biochemical signaling cascades. In the following sections, we focus on the LINC complex as the central mediator of this direct mechanical transmission pathway.

Actin-nesprin interactions within the LINC complex

As the primary interface through which cytoskeletal forces reach the nucleus, the LINC complex relies on nesprins to establish mechanical coupling with distinct cytoskeletal filament systems. Nesprins contain a conserved C-terminal Klarsicht, ANC-1, and Syne homology (KASH) domain that inserts into the ONM and anchors the protein to the nuclear surface, while their extended N-terminal regions project into the cytoplasm to engage cytoskeletal components. ²²

Filament specificity is determined by nesprin isoform composition. Nesprin-1 and nesprin-2, the largest isoforms, harbor calponin homology domains that directly bind filamentous actin, enabling efficient transmission of actomyosin-generated tension to the nuclear envelope. In contrast, microtubule-dependent connections are largely indirect: nesprin-1 and nesprin-2 recruit dynein–dynactin complexes, whereas shorter isoforms such as nesprin-1α and nesprin-4 associate with kinesin motors to facilitate nuclear positioning and transport. For intermediate filaments, nesprin-3 links the nucleus to the cytoskeleton through the cytolinker plectin. ¹⁰ Collectively, these isoform-specific interactions allow the LINC complex to integrate tensile, compressive, and transport-related forces from multiple cytoskeletal networks, thereby ensuring robust and versatile mechanical coupling between the cytoplasm and the nucleus.

Nesprin-SUN interactions within the LINC complex

As forces propagate across the nuclear envelope, SUN proteins serve as the central transmembrane relay that transfers nesprin-mediated tension between the two nuclear membranes. Mammalian cells predominantly express SUN1 and SUN2, which oligomerize through extended coiled-coil domains to form stable trimeric assemblies. ²⁶ This trimeric architecture provides mechanical reinforcement and distributes force across multiple binding interfaces, thereby enhancing the stability of the complex under sustained stress. The C-terminal SUN domains project into the perinuclear space, where they bind directly to the KASH domains of nesprins. ²⁷ This SUN–KASH interaction mechanically couples the ONM and INM, forming a continuous transmembrane bridge that enables cytoskeletal forces to traverse the nuclear envelope without membrane rupture. Functionally, this interface acts as a molecular “clutch,” ensuring efficient force transmission while preserving structural integrity during processes such as migration, confinement, and tissue deformation.

SUN-nucleoskeleton interactions within the LINC complex

Once mechanical forces cross the nuclear envelope, the SUN trimeric complex transmits them directly to the nucleoskeleton. The N-terminal transmembrane segments anchor SUN proteins within the INM, while adjacent lamin-binding domains interact with lamin A/C and associated lamina components.^28,29 Through these connections, forces delivered from the cytoskeleton are distributed across the nuclear lamina and the underlying chromatin network. This coupling integrates mechanical strain into the structural framework of the nucleus, influencing nuclear stiffness, chromatin tethering, and higher-order genome organization. Consequently, physical forces are not merely transmitted to the nuclear periphery but are propagated into the nuclear interior, where they directly modulate chromatin architecture and gene regulatory landscapes. In this manner, the SUN–lamin interface serves as a critical mechanotransduction hub that links cytoskeletal tension to transcriptional control.

Nuclear deformation and structural mechanosensing

Mechanical forces delivered to the nucleus through the LINC complex do not merely reposition the organelle within the cytoplasm; rather, they directly deform the nuclear envelope and remodel nuclear architecture. Such deformation alters membrane tension, curvature, and lamina strain, generating physical stresses that propagate across the nuclear surface and into the underlying nucleoskeleton. These structural perturbations influence the conformation, localization, and activity of multiple mechanosensitive proteins embedded in or associated with the nuclear envelope.

Through these mechanically induced changes, the nucleus functions simultaneously as a force sensor and a signal transducer. Increased nuclear envelope tension can modulate nuclear pore permeability, reorganize inner nuclear membrane protein–chromatin interactions,^30,31 and remodel the lamin network through post-translational modifications, including lamin A/C phosphorylation and assembly–disassembly dynamics. ³² Collectively, these responses convert physical deformation into biochemical and transcriptional outputs by regulating nucleocytoplasmic transport, chromatin accessibility, and nuclear stiffness. Thus, the nucleus operates as an active mechanosensitive organelle that directly translates mechanical inputs into gene regulatory consequences rather than serving as a passive structural compartment.

Nuclear pore complex

The nuclear pore complex (NPC) represents a key interface through which nuclear mechanics are coupled to intracellular signaling. NPCs are ∼100-nm-diameter macromolecular assemblies embedded within the nuclear envelope and consist of a structural scaffold, a central transport channel, cytoplasmic filaments, and a nuclear basket. Selective transport is governed by a meshwork of phenylalanine–glycine-rich nucleoporins (FG-Nups) that forms a dynamic diffusion barrier, thereby regulating bidirectional trafficking of proteins, RNA, and signaling molecules between the nucleus and cytoplasm.^33,34 Emerging evidence indicates that NPCs are intrinsically mechanosensitive structures. Elevated nuclear envelope tension can physically dilate the pore scaffold and reduce the effective density of FG-Nups, weakening the selectivity barrier and facilitating macromolecular transport. Under these conditions, nuclear import of transcription factors, kinases, and regulatory RNAs is enhanced, enabling rapid activation of downstream signaling pathways and transcriptional programs. Conversely, relaxation of envelope tension restores barrier selectivity and restricts molecular flux, thereby dampening nuclear signaling.^33,35 Accordingly, NPCs function not only as transport conduits but also as mechanically gated channels that dynamically couple nuclear deformation to nucleocytoplasmic transport kinetics. This gating mechanism provides a rapid and reversible route through which physical forces directly modulate transcriptional responses.

Interactions with chromatin and LEMD family proteins

Inner nuclear membrane proteins of the LAP2–emerin–MAN1 domain (LEMD) family—including lamina-associated polypeptide 2 (LAP2), emerin, MAN1/LEMD3, and LEMD2—serve as key mediators linking the nuclear envelope to chromatin. These proteins interact with lamina-associated domains (LADs) through the DNA-binding protein Barrier-to-Autointegration Factor (BAF), forming LEMD–BAF–LAD complexes that tether chromatin to the nuclear periphery and generally promote transcriptional repression.^36,37 Mechanical forces that alter nuclear envelope tension modulate the efficiency of this tethering system. Changes in LEMD protein localization, phosphorylation status, and affinity for lamins influence BAF-mediated chromatin anchoring. ³⁸ Consequently, subsets of LADs may detach from the lamina and reposition toward the nuclear interior, whereas others become more stably anchored at the periphery. Regions that remain tightly tethered typically exhibit increased chromatin compaction, reduced DNA accessibility, and transcriptional repression, whereas weakly tethered or internalized regions display chromatin decondensation, enhanced accessibility, and gene activation.^39,40 These spatial rearrangements are frequently accompanied by changes in histone post-translational modifications (PTMs), reinforcing epigenetic remodeling in response to mechanical cues.

In addition to LEMD proteins, lamina-associated polypeptide 1 (LAP1) contributes to nuclear envelope integrity and lamin-dependent nuclear mechanics. Unlike LAP2, LAP1 lacks a LEM domain and does not directly form chromatin-associated complexes. ⁴¹ Instead, its N-terminal region forms stable interactions with lamin B and associates with lamin A/C and emerin, thereby reinforcing structural coupling between the inner nuclear membrane and the nuclear lamina. ⁴² Consistent with this architectural role, LAP1 deficiency increases nuclear deformability under compressive stress and frequently results in nuclear envelope blebbing. ⁴³ Thus, although LAP1 does not directly tether chromatin, it indirectly influences chromatin organization by modulating the transmission and distribution of mechanical forces across the nuclear envelope.

Nuclear lamina

The nuclear lamina, composed primarily of lamin A/C and lamin B isoforms (B1 and B2), forms a filamentous meshwork underlying the inner nuclear membrane that provides structural support while serving as a central hub for nuclear mechanosensing. ⁴⁴ Beyond acting as a passive scaffold, the lamina dynamically adjusts its composition and mechanical properties in response to extracellular stiffness and cytoskeletal tension. Among lamins, lamin A/C exhibits pronounced mechanosensitivity. On stiffer extracellular matrices, phosphorylation of lamin A/C at Ser22 decreases, reducing turnover and proteolytic degradation, thereby increasing steady-state lamin levels. ⁴⁵ This accumulation stiffens the nuclear envelope and enhances resistance to deformation. In mechanically active tissues such as skeletal and cardiac muscle, this stiffening protects the nucleus from repetitive strain and stabilizes lamina-associated domain (LAD) tethering, contributing to sustained chromatin compaction and long-term repression of specific gene programs. ⁴⁶ Accordingly, lamin A/C functions as an adaptive mechanical rheostat that tunes nuclear stiffness to match environmental demands.

In contrast, lamin B isoforms are more uniformly distributed and act as constitutive structural scaffolds essential for nuclear integrity, cell division, and survival. Lamin B associates strongly with peripheral heterochromatin through proteins such as the lamin B receptor (LBR), thereby maintaining chromatin organization at the nuclear periphery. ⁴⁷ This lamin B network also preserves nuclear shape and stabilizes NPC architecture under mechanical stress. Disruption or weakening of either lamin A/C or lamin B networks compromises nuclear mechanical resilience. Under excessive strain, such defects can concentrate stress at vulnerable regions of the envelope, leading to nuclear envelope rupture, chromatin herniation, and activation of DNA damage responses. ⁴⁸ These events not only threaten genome integrity but also trigger downstream signaling pathways that alter cell fate and transcriptional programs. Collectively, the nuclear lamina operates as both a structural framework and a mechanotransductive platform that integrates mechanical inputs with chromatin organization and gene regulation. By modulating nuclear stiffness, envelope stability, and chromatin mobility, lamin networks directly shape the sensitivity of the nucleus to mechanical cues and thereby influence diverse cellular signaling and transcriptional outcomes.

Transcriptional mechanotransduction and mechanosensitive nuclear signaling

Mechanical forces transmitted to the nuclear envelope, lamina, and chromatin ultimately converge on transcriptional regulation through coordinated epigenetic remodeling and mechanosensitive signaling pathways. Changes in nuclear tension and architecture reorganize lamina–LEMD–heterochromatin interactions, which in turn influence the localization and activity of histone-modifying enzymes and chromatin regulators. These alterations reshape patterns of histone acetylation and methylation, thereby modulating chromatin accessibility and selectively tuning transcriptional competence.^40,49 Concurrently, mechanical inputs regulate nucleocytoplasmic transport and cytoskeletal dynamics. Variations in nuclear shape, NPC permeability, and actomyosin contractility interface with the Hippo and RhoA/ROCK pathways to control the phosphorylation state and nuclear–cytoplasmic shuttling of mechanosensitive transcription factors, including YAP/TAZ–TEAD complexes and myocardin-related transcription factor A (MKL1)/serum response factor (SRF).^50,51 These mechanosensitive pathways are also operative in pathological contexts; in pancreatic β-cells, diabetes-associated ECM stiffening activates the Hippo-YAP/TAZ axis, thereby dysregulating β-cell proliferation and phenotypic stability. ⁵² Through these parallel mechanisms, the nucleus integrates physical cues into coordinated transcriptional programs that adapt cellular behavior to the mechanical microenvironment.

Histone modification

Mechanical cues directly influence chromatin organization by regulating the activity of histone-modifying enzymes. Changes in substrate stiffness, shear stress, hydrostatic pressure, and tensile or compressive forces collectively alter the balance between histone acetylation and deacetylation, thereby modifying transcriptional accessibility.^53,54 Among histone deacetylases (HDACs), HDAC3 is particularly responsive to mechanical stimulation. Increased nuclear envelope tension or matrix stiffness promotes its association with the nuclear lamina and emerin, facilitating deacetylation of genes located near lamina-associated domains (LADs) and reinforcing transcriptional repression.^55,56 In endothelial cells, inhibition of HDAC activity with trichostatin A under shear stress elevates histone H3 acetylation and phosphorylation, further supporting the mechanosensitivity of chromatin remodeling enzymes. ⁵⁷ Conversely, histone acetyltransferases (HATs) can also be activated by mechanical inputs. For example, p300 translocates to the nucleus in hepatic stellate cells exposed to increased matrix stiffness, enhancing H3K27 acetylation and promoting transcription of stiffness-responsive genes such as CXCL12. ⁵⁸ Together, these opposing HDAC and HAT activities dynamically regulate acetylation landscapes in response to physical forces. ⁵⁹ Histone methylation is likewise mechanoregulated. Enhancer of zeste homolog 2 (EZH2), the catalytic component of Polycomb repressive complex 2, decreases in expression under abnormal shear stress or altered matrix stiffness, leading to reduced H3K27me3 deposition and changes in gene repression. ⁵⁴ Nuclear softening is also associated with loss of H3K9me3, a hallmark of compact heterochromatin. ³⁰ Collectively, these findings demonstrate that histone modifications provide a molecular interface through which mechanical signals are encoded into stable transcriptional outcomes.

Mechanosensitive control of transcription factors

Beyond chromatin remodeling, mechanical stimuli regulate transcription more directly by controlling the activity and localization of mechanosensitive transcription factors. A central pathway in this process is Hippo signaling, mediated by the upstream kinases mammalian STE20-like protein kinase 1/2 (MST1/2) and large tumor suppressor homolog 1/2 (LATS1/2). On soft substrates or under low cytoskeletal tension, Hippo signaling is activated, resulting in phosphorylation of YAP and TAZ by MST1/2–LATS1/2. Phosphorylated YAP/TAZ bind 14-3-3 proteins, leading to cytoplasmic retention or degradation and suppression of transcriptional activity.^60–62 In contrast, on stiff substrates that promote actomyosin contractility, Hippo–LATS activity is inhibited, reducing YAP/TAZ phosphorylation and allowing them to remain in an unphosphorylated, active state. Under these conditions, increased nuclear tension and enhanced NPC permeability further facilitate nuclear import and accumulation of YAP/TAZ.^63,64 Once inside the nucleus, YAP/TAZ associate with TEAD transcription factors to activate gene programs controlling proliferation, survival, and extracellular matrix remodeling.^63,65 Similarly, MKL1 responds to actin polymerization dynamics: increased F-actin levels release MKL1 from G-actin sequestration, enabling its nuclear translocation, where it cooperates with SRF to induce expression of cytoskeletal and contractile genes. ⁶⁶ Through these complementary mechanisms, mechanical forces regulate both epigenetic states and transcription factor availability, ensuring that gene expression programs are tightly coupled to the physical properties of the cellular environment. ⁶⁷

Using FRET-based techniques to monitor nuclear mechanotransduction

Mechanical stimuli transmitted from the cell surface to the nucleus evoke rapid structural, biochemical, and signaling responses that often occur on sub-second to minute timescales and involve nanoscale molecular rearrangements. Accordingly, experimental approaches capable of detecting fast conformational changes and dynamic protein interactions are essential for dissecting nuclear mechanotransduction in living cells.

Fluorescence resonance energy transfer (FRET), which sensitively reports intermolecular proximity and conformational shifts at nanometer resolution, has therefore emerged as a particularly powerful strategy for studying nuclear mechanics in real time. By converting distance-dependent energy transfer into quantifiable fluorescence changes, FRET enables direct visualization of protein stretching, strain, and transient molecular interactions within intact cells. In the following sections, we describe how genetically encoded FRET biosensors and FRET-based imaging approaches have been applied to monitor conformational changes, molecular tension, and protein–protein interactions within key components of the nuclear force transmission machinery. Representative studies employing these strategies are summarized in Figure 1 and Table 1 together with their mechanistic insights.

Table 1.

FRET-based biosensors and assays for nuclear mechanotransduction.

Mechanotransductive stage	Sensor target	Detectable signal	Sensing module	Sensing principle	Readout	FP pair		Ref.
						Donor	Acceptor
Direct mechanical transduction	LINC Complex	Tension on Nesprin-2G	Actin Binding Domain, elastic linker, SUN Binding Domain	Low FRET (High tension)	FRET	mTFP1	Venus	68
			Calponin homology domain, TM KASH domain	Low FRET (High tension)	FRET	mTFP1	EYFP	72
			Calponin homology domain (include spectrin repeat region), TM KASH domain	Low FRET (High tension)	FRET	mTFP1	Venus	73
Mechanically induced Nuclear deformation	Nuclear Lamina	Strain on Lamin A/C	Lamin Chromobody	Low FRET (High strain)	FRET	mTFP1	mVenus	76
	LEM and BAF	BAF-Emerin interaction	BAF, Emerin	High FRET (BAF-Emerin interaction)	FRET (Acceptor photobleaching)	CFP	YFP	77
		BAF interaction	BAF	High FRET (BAF assemble)	FRET (Acceptor photobleaching)	mCFP	mVenus	78
		LAP1-Emerin interaction	LAP1, Emerin	High FRET (LAP1-Emerin interaction)	FRET (Acceptor photobleaching)	GFP	RFP	79
Transcriptional mechanotransduction	Histone Epigenetic Modification	Histone Compaction	Histone H2B	High FRET (Chromatin compaction)	FLIM-FRET	GFP	mCherry	80
		Histone Phosphorylation	Histone H3 peptide, 14-3-3τ	High FRET (Phosphorylation)	FRET	CFP	YFP	81
		Histone Methylation	Histone H3 peptide, Chromodomain (HP1 or Pc)	High FRET (Methylation)	FRET	CFP	YFP	82
			Histone H3, Chromodomain, Centromeric DNA-binding domain (CENP-B)	High FRET (Centromeric methylation)	FRET	Cerulean	Venus	83
			Histone H3, Chromodomain (HP1)	High FRET (Methylation)	FRET	ECFP	YPet	84
		Histone Acetylation	Histone H4 or H1, Bromodomain (BRD2)	High FRET (Acetylation)	FC-FRET	CFP	YFP	85
			Histone H4, Bromodomain testis-specific protein (BRDT)	Low FRET (Acetylation)	FRET	CFP	Venus	86
			Histone H4, Bromodomain (BRD2)	Low FRET (Acetylation)	FRET	CFP	Venus	87
			Histone H3, Bromodomain (BRD4)	Low FRET (Acetylation)	FRET	CFP	YFP	88
			Histone H3, ScFv intrabody (VH and VL)	High FRET (Acetylation)	FRET	SeCFPdC5	YPet	89
			Histone H4 peptide, Bromodomain	Low FRET (Acetylation)	FRET	CFP	YFP	90
	YAP Localization	Angiomotin (AMOT)-YAP interaction	AMOT, YAP	High FRET (AMOT-YAP interaction)	FRET (Acceptor photobleaching)	CFP	YFP	91
		YAP-p73 interaction	YAP, p73	High FRET (p73-YAP interaction)	FRET	GFP	YFP	92
		YAP-14-3-3 interaction	YAP, 14-3-3	High FRET (14-3-3-YAP interaction)	FRET	DiB1	mNeonGreen	93

Abbreviations: FLIM-FRET, Fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer; FC-FRET, Flow cytometry-based fluorescence resonance energy transfer.

Monitoring direct mechanical transduction

Nesprin (LINC complex) interaction

Mechanical forces generated within the cytoskeleton are transmitted to the nucleus primarily through the LINC complex, with nesprins serving as critical mechanical linkers that directly couple cytoskeletal tension to the nuclear envelope. Because nesprins undergo force-dependent extension and compression, they represent attractive targets for FRET-based tension sensing. Arsenovic et al. engineered a genetically encoded nesprin tension sensor by inserting a donor–acceptor FRET pair connected by an elastic linker between the actin-binding domain and the SUN-binding domain of nesprin. ⁶⁸ Changes in FRET efficiency reported force-induced molecular stretching, providing direct optical evidence that cytoskeletal contractility physically elongates the LINC complex. This work established the first demonstration that mechanical tension is transmitted across the nuclear envelope and positioned nesprin-based FRET sensors as foundational tools in nuclear mechanobiology. Subsequent studies extended this platform to diverse biological contexts. Using the same sensor, Lee et al. showed that a specific microRNA regulates lamin A/C expression and thereby modulates nuclear mechanosensitivity. ⁶⁹ Sohn et al. applied the sensor to uncover a mechanistic link between tau pathology and altered nuclear tension in neurons. ⁷⁰ Fenelon et al. further demonstrated that forces acting on the nuclear membrane can be quantified in vivo within intact tissues. ⁷¹ Additional nesprin-based FRET sensor variants have refined understanding of domain-specific mechanics. Dejardin et al. constructed a sensor spanning the calponin homology (CH) and KASH domains and demonstrated that nesprin-mediated tension contributes to epithelial–mesenchymal transition (EMT). ⁷² Carley et al. developed a sensor incorporating the CH domain and spectrin-repeat regions, revealing that LINC-dependent forces regulate epidermal differentiation. ⁷³ Collectively, these studies establish nesprin tension sensors as robust tools for directly visualizing force transmission across the nuclear envelope.

Monitoring mechanically induced nuclear deformation

Nuclear lamina modification

Mechanical deformation of the nucleus not only stretches the LINC complex but also induces strain within the lamin polymer network. The nuclear lamina undergoes characteristic rearrangements, including filament displacement, reorganization, and strain-dependent structural remodeling, which together determine nuclear stiffness and mechanical resilience.^74,75 However, these nanoscale alterations are difficult to resolve using conventional imaging approaches.

To directly monitor lamina mechanics, Danielsson et al. developed a FRET-based lamina strain biosensor by positioning fluorescent donor and acceptor proteins within lamin-targeting chromobodies. ⁷⁶ This design enables FRET efficiency to change in response to compression or extension of lamin filaments, providing a real-time optical readout of lamina strain. Using this approach, the authors demonstrated that mechanical forces dynamically reshape lamin organization and directly influence nuclear structural integrity. ⁷³ These findings illustrate how FRET-based lamina sensors complement nesprin tension probes by reporting mechanical responses downstream of force transmission, thereby enabling quantitative visualization of how cytoskeletal forces propagate from the nuclear surface into the nuclear interior.

LEM-domain proteins and BAF protein dynamics

FRET-based approaches have also been instrumental in resolving the dynamic interactions between Barrier-to-Autointegration Factor (BAF) and LEM-domain proteins at the nuclear envelope. Because these proteins form the molecular bridge linking the nuclear lamina to peripheral chromatin, their spatiotemporal organization is critical for mechanically regulated chromatin tethering and nuclear architecture. However, these interactions are highly dynamic and difficult to capture using conventional biochemical assays. Live-cell FRET imaging has enabled direct visualization of these transient associations. Shimi et al. demonstrated a direct interaction between BAF and emerin using CFP–BAF and YFP–emerin fusion constructs, revealing that their binding strength fluctuates throughout the cell cycle. ⁷⁷ Haraguchi et al. further showed that BAF undergoes rapid redistribution during mitosis, supporting its essential role in nuclear envelope breakdown and reassembly. ⁷⁸ Beyond BAF–emerin interactions, FRET has also been used to probe interactions among LEM-domain proteins themselves. Shin et al. employed FRET analysis between RFP–emerin and GFP–LAP1 to identify dynamic binding patterns within the LEM network, uncovering functional coupling between emerin–LAP1 interactions and nuclear structural integrity. ⁷⁹ Collectively, these studies demonstrate that FRET provides a powerful means to monitor the temporal and spatial regulation of LEM–BAF complexes that mediate chromatin–lamina coupling during nuclear mechanotransduction.

Monitoring transcriptional mechanotransduction

Histone epigenetic modification

FRET-based imaging has been widely applied to investigate chromatin remodeling and histone modifications with high spatiotemporal resolution, providing critical insights into how mechanical cues are translated into epigenetic changes. By reporting local chromatin compaction or modification-dependent protein interactions, these sensors enable direct visualization of nuclear states that were previously accessible only through fixed-cell or population-based assays. Early studies by Llères et al. fused GFP and mCherry to histone H2B to generate an intranucleosomal FRET reporter of chromatin compaction, revealing stage-specific condensation states during mitosis. ⁸⁰ Building on this strategy, Lin et al. developed genetically encoded FRET biosensors to monitor histone phosphorylation (H3S28p) and methylation marks such as H3K9me3 and H3K27me3 in living cells.^81,82 Subsequent work refined these tools for locus-specific or modification-specific readouts. Chu et al. applied a centromere-targeted H3K9me3 biosensor to map methylation dynamics during mitosis, whereas Peng et al. developed an H3K9me3 sensor exhibiting anticorrelated behavior with H3S10 phosphorylation, illustrating coordinated regulation between methylation and phosphorylation events.^83,84 FRET has also been extensively used to probe histone acetylation. Kanno et al. visualized acetylation-dependent interactions using CFP–Brd2 and YFP–H4. ⁸⁵ Sasaki et al. engineered a bromodomain testis-specific protein (BRDT)–based sensor to monitor H4K5 and H4K8 acetylation. ⁸⁶ Ito et al. subsequently targeted H4K12 acetylation, ⁸⁷ and Nakaoka et al. expanded this approach to H3K9 and H3K14 acetylation. ⁸⁸ More recently, Chung et al. introduced an intrabody-based FRET probe enabling visualization of endogenous H3K9 acetylation, ⁸⁹ and Han et al. developed a biosensor reporting histone H4 N-terminal acetylation with high sensitivity. ⁹⁰ Together, these studies highlight the versatility of FRET for monitoring dynamic epigenetic states in living cells and provide valuable tools for linking mechanical perturbations to chromatin-level regulation.

YAP localization

FRET-based strategies have likewise provided important insights into the dynamic regulation of mechanosensitive transcription factors such as Yes-associated protein (YAP). Because YAP activity is strongly governed by its subcellular localization and interaction partners, real-time visualization of its molecular associations is essential for understanding mechanotransduction signaling. Zhao et al. used FRET imaging to demonstrate that angiomotin (AMOT) directly interacts with YAP and restricts its nuclear localization, thereby suppressing YAP-dependent transcription. ⁹¹ Zhang et al. further showed that YAP associates with p73 during amyloid-β peptide (Aβ₂₅–₃₅)-induced apoptosis, revealing a functional connection between YAP signaling and stress-induced cell death pathways. ⁹² More recently, Gavrikov et al. introduced a simplified single-channel FRET strategy using the fluorogen-activating protein DiB1, enabling visualization of both YAP localization and YAP–14-3-3 interactions with reduced optical complexity and improved imaging practicality. ⁹³ Together, these studies demonstrate that FRET provides a versatile platform for dissecting the spatial redistribution and interaction dynamics of YAP, thereby linking mechanical signals to downstream transcriptional regulation.

Conclusion

In this review, we integrated current knowledge of both direct and indirect pathways governing nuclear mechanotransduction and highlighted FRET-based genetically encoded biosensors as powerful tools for interrogating these processes in living cells. Mechanical cues originating at the cell surface are transmitted through the cytoskeleton and nuclear envelope, inducing nuclear deformation and reorganizing nuclear envelope proteins, lamina networks, and chromatin architecture. These structural changes ultimately reshape transcriptional programs, positioning the nucleus as both a mechanical sensor and an active regulator of gene expression.

As mechanotransduction has emerged as a fundamental determinant of cell fate, development, and disease, experimental platforms such as Organ-on-a-Chip systems, atomic force microscopy, and traction force microscopy have enabled increasingly precise manipulation of extracellular mechanical environments.^94–96 However, approaches for resolving the intracellular molecular events triggered by these forces—particularly within the nucleus—remain comparatively limited. For instance, traction force microscopy can be influenced by analysis sensitivity and substrate properties, which in some contexts may complicate interpretation of cellular mechanoadaptive responses. ⁹⁷ In this context, FRET-GEBs provide a complementary advantage by enabling continuous, real-time visualization of stimulus–response dynamics while preserving the native mechanical context of living cells.

Mechanotransduction is transmitted through physical coupling across complex intracellular architectures rather than through chemical diffusion. Accordingly, FRET biosensors designed to monitor mechanotransduction are often targeted to force-bearing structures, including focal adhesions, the nuclear envelope, and chromatin. However, these structural microenvironments can influence the basal signal of FRET biosensors in multiple ways.

First, owing to the structural complexity of these compartments, FRET sensors may not maintain sufficient separation between the donor and acceptor fluorophores. In addition, high molecular crowding—particularly within the nucleus, where chromatin and nucleoproteins are densely packed—can bias FRET readouts.^98,99 To address these challenges, D. Llères et al. applied fluorescence lifetime imaging microscopy (FLIM)–FRET in chromatin-condensed nuclei and improved measurement reliability through background thresholding, χ²-guided fitting, and donor-only correction. ⁸⁰

Second, changes in refractive index associated with the physical condensation of biomolecules can also affect FRET signals. According to the Strickler and Berg equation, the fluorescence lifetime of fluorophores and fluorescent proteins is inversely proportional to the square of the refractive index.^100,101 Thus, as the refractive index increases, the radiative emission rate also increases, potentially altering both the dynamic range and the basal apparent FRET efficiency of biosensors. ¹⁰² Moreover, the intracellular refractive index is not spatially uniform across subcellular compartments and has been reported to vary depending on cell line and cell-cycle stage. ¹⁰³ In particular, the nucleus generally exhibits a higher refractive index than the cytoplasm. ¹⁰⁴ In LoVo cells, the refractive index is higher in G2 phase than in G1 phase, consistent with increased chromatin content during G2. ¹⁰⁵ In NIH3T3 cells, heterochromatin has been reported to be approximately 1.53-fold denser than euchromatin, corresponding to a higher refractive index. ¹⁰⁶

Nevertheless, some studies have turned these apparent limitations into informative readouts. For instance, S. M. Levchenko et al. used FLIM–FRET to measure donor fluorescence lifetimes and infer DNA compaction from lifetime shifts reflecting local refractive-index changes associated with variations in DNA and protein concentration. ¹⁰⁷ In addition, FRET sensors that directly report molecular crowding have also been developed.^108–111 Taken together, continued investigation of the factors that modulate FRET signals, along with their biological implications, may facilitate the development of new FRET sensors specifically designed to detect these parameters. This perspective suggests that features once regarded as experimental limitations may instead be repurposed as informative readouts that provide new biological insight.

Although current FRET-based approaches face technical challenges, including photobleaching, spectral crosstalk, and limited multiplexing,^112,113 additional constraints remain. Because FRET efficiency depends on both inter-fluorophore distance and dipole orientation (k ² ), force directionality cannot be determined from a single FRET readout. Although polarization-resolved imaging offers a partial solution, it requires complex optical instrumentation, provides reduced spatial resolution, and has not yet been applied to genetically encoded intranuclear sensors. ¹¹⁴ Furthermore, expression heterogeneity and delivery limitations in primary and immune cells restrict broader applicability, although electroporation of purified biosensors and transposon-mediated stable integration represent promising alternatives.^115,116 Encouragingly, integration with FLIM, ¹¹⁷ next-generation fluorescent proteins, and AI-assisted image analysis is rapidly mitigating these constraints. ¹¹⁸ These advances are expected to substantially improve sensitivity, quantitative robustness, and throughput, expanding nuclear mechanobiology from single-pathway measurements toward multiplexed and systems-level analyses. Despite being less developed than tools targeting ECM–cytoskeleton mechanotransduction, FRET-based strategies focused on nuclear processes hold exceptional promise for elucidating how mechanical forces regulate lamina mechanics, nucleocytoplasmic transport, chromatin organization, and transcriptional control. By categorizing mechanical stimuli and distilling common design principles across existing sensors, this review provides a framework for the rational development of next-generation FRET-GEBs.

Collectively, continued innovation in genetically encoded biosensors will be essential for bridging nuclear mechanics and gene regulation. We anticipate that these tools will enable a more quantitative and mechanistic understanding of how physical forces shape genome function and will accelerate the integration of mechanobiology into both fundamental research and translational applications.