Lamins at the crossroads of mechanosignaling

Assembly and mechanical properties of lamins: lamins as ‘shock absorbers’

One important property of nuclear lamins, essential for most if not all of their cellular functions, is their ability to assemble into higher-order structures. Lamin assembly has been studied intensively in vitro, but there are still a lot of open questions regarding their higher-order structure organization in vivo (Zwerger and Medalia 2013). The basic subunit of lamin assembly is a parallel coiled-coil dimer (Aebi et al. 1986; Dessev et al. 1990; Gieffers and Krohne 1991; Heitlinger et al. 1991; Stuurman et al. 1996). In vitro, these dimers assemble “head to tail” to form polar filaments, which interact laterally in an anti-parallel fashion to form apolar tetrameric protofilaments. These assemble into paracrystals (Stuurman et al. 1998; Zwerger and Medalia 2013) or, in the case of Caenorhabditis elegans, also into ∼10-nm-thick filamentous structures (three to four protofilaments) (Karabinos et al. 2003; Foeger et al. 2006; Ben-Harush et al. 2009).

In contrast, little is known about the assembly of lamins into the nuclear lamina in vivo. Lamins are extensively modified and interact with a large number of proteins (Simon and Wilson 2011; Korfali et al. 2012), the nuclear membrane, and chromatin (Kalinowski et al. 2013; Simon and Wilson 2013), all of which can affect lamin assembly.

For example, lamin A and all B-type lamins contain a −CAAX box at their C-terminal end, which leads to their extensive post-translational processing. In a first step, a farnesyl moiety is attached to the cystein residue by a farnesyltransferase, and then the −AAX tripeptide is cleaved off by an endopeptidase, most likely Ras-converting enzyme 1 (Rce1) and/or zinc metalloprotease related to Ste24p/FACE1 (Zmpste24); subsequently, the cysteine residue is carboxymethylated by isoprenylcystein carboxyl methyltransferase (Icmt) (Young et al. 2005; Rusinol and Sinensky 2006). While the processing of B-type lamins ends here, an additional 15 amino acids, including the farnesylated/carboxymethylated cysteine residue, are cleaved off of the C terminus of lamin A in a final step, leading to its mature form (Bergo et al. 2002; Pendas et al. 2002; Corrigan et al. 2005). Therefore, mature B-type lamins contain a farnesyl and carboxymethyl group at their C termini, while mature lamin A and lamin C, which do not contain a −CAAX box, lack such modifications. The −CAAX processing occurs most likely within the nucleus (Barrowman et al. 2008) and is suggested to be involved in targeting lamins to the INM and establishing protein–protein interactions (Rusinol and Sinensky 2006; Kalinowski et al. 2013). Although the permanent farnesylation/carboxymethylation of lamins leads to their stable association with membranes throughout the cell cycle, it is not a prerequisite for their incorporation into the nuclear lamina (Dechat et al. 2007, 2008).

In addition to farnesylation, lamins become extensively phosphorylated during the cell cycle (Simon and Wilson 2013; Kochin et al. 2014). Lamin phosphorylation was initially described in mitosis, where it leads to the depolymerization and solubilization of lamin structures required for nuclear envelope (NE) breakdown (Gerace and Blobel 1980; Heald and McKeon 1990; Collas 1999; Panorchan et al. 2004a; Mall et al. 2012). Upon NE reassembly at the end of mitosis, lamin dephosphorylation is required for the formation of a nuclear lamina (Thompson et al. 1997; Steen et al. 2000; Ito et al. 2007). While B-type lamins relocate exclusively to the periphery of the newly forming sister nuclei, A-type lamins initially accumulate within the nuclear interior during telophase and early G1 (Moir et al. 2000; Dechat et al. 2004). Besides their mitotic phosphorylation, lamins become also phosphorylated during interphase, which appears to be important for the regulation of their nuclear import and their solubility (Hennekes et al. 1993; Schneider et al. 1999; Cenni et al. 2005; Kuga et al. 2010; Mitsuhashi et al. 2010; Zaremba-Czogalla et al. 2012; Buxboim et al. 2014; Kochin et al. 2014).

The molecular and structural organization of the B- and A-type lamins in the nuclear lamina is still not completely understood. For many years the lamina was thought to represent an orthogonal meshwork of 10-nm filaments, based on early electron microscopic studies in Xenopus oocyte nuclei (Aebi et al. 1986). However, such a network has not been seen in somatic cells so far. Based on the ability of lamins to form heterodimers in vitro (Schirmer et al. 2001), it was assumed that B- and A-type lamins can form heteropolymers also in vivo. However several recent studies, including fluorescence resonance energy transfer and superresolution microscopy analyses, and the expression of various lamin isoforms in Xenopus oocyte nuclei clearly revealed independent but interconnected networks of A- and B-type lamins (Goldberg et al. 2008; Schermelleh et al. 2008; Shimi et al. 2008; Kolb et al. 2011; Grossman et al. 2012). These data support a model in which the farnesylated B-type lamins, especially lamin B1, form a more regular network closely associated with the INM and the nuclear pore complexes (NPCs) while the A-type lamins form more irregular meshworks on top of the lamin B structures. This model is supported by the findings that knockdown of lamin B1 results in a dramatic increase in the mesh size of the lamin A/C and lamin B2 networks and leads to the formation of nuclear blebs enriched in A-type lamins and deficient for B-type lamins (Shimi et al. 2008). Furthermore, studies in ESCs lacking different combinations of B- and A-type lamins and in fibroblast-like cells derived therefrom also showed that lamin A/C and lamin B2 structures are dependent on lamin B1 (Guo et al. 2014), but, at sufficiently high concentration, all lamins can form a lamina on their own.

A-type lamins are also present within the nuclear interior in interphase cells, most likely due to the lack of a C-terminal farnesyl group. These nucleoplasmic lamins A/C are most prominent in early G1 cells, more dynamic than the lamins residing in the lamina, and regulated through phosphorylation and interaction with the lamina-associated polypeptide 2α (LAP2α) (Dechat et al. 2000, 2004; Moir et al. 2000; Naetar et al. 2008; Shimi et al. 2008; Naetar and Foisner 2009; Kolb et al. 2011; Kochin et al. 2014). Although little is known about the regulation, dynamics, and assembly state of the nucleoplasmic A-type lamins, they may be involved in a variety of cellular processes, including chromatin organization, gene expression, and cell proliferation (Dechat et al. 2010b; Gesson et al. 2014).

Recent studies have shown that lamins can form stiff yet elastic and compressible networks suggested to function as “molecular shock absorbers” (Dahl et al. 2004, 2005; Panorchan et al. 2004b). The mechanical properties of the nucleus are highly dependent on the molecular and structural composition of the nuclear lamina. For example, nuclear mechanics is impaired in lamin A/C-deficient cells and cells devoid of emerin, an integral protein of the INM that interacts with lamins (Broers et al. 2004; Lammerding et al. 2004, 2005; Rowat et al. 2006; Lee et al. 2007). Furthermore, it appears that the mechanical stiffness of the nucleus and its viscosity are solely dependent on A-type but not B-type lamins, while B-type lamins provide elasticity to the nucleus and the ability to deform. Thus, cells expressing very low levels of lamins A and C such as ESCs display a high degree of nuclear plasticity (Lammerding et al. 2006; Pajerowski et al. 2007). Accordingly, cells expressing high levels of lamins A and C have a high nuclear stiffness, which impairs the cells’ ability to migrate through constraining micropores (Rowat et al. 2013; Shin et al. 2013; Harada et al. 2014).

Lamins in signaling and gene expression

Lamins affect a number of signaling pathways, some of which are tissue-specific (Heessen and Fornerod 2007; Andres and Gonzalez 2009). Their mode of action can be explained by forming a scaffolding structure that interacts with and regulates signaling molecules and transcription factors. On the one hand, lamins can attenuate signaling pathways by acting as a “peripheral nuclear trap” for transcriptional regulators, such as c-Fos and a transcriptional coactivator of the Notch pathway (SKIP) (Ivorra et al. 2006; Scaffidi and Misteli 2008). On the other hand, lamins can serve as a platform for signaling molecules, enabling efficient reactions. For example, phosphorylation of c-Fos by the extracellular signal-regulated kinase (ERK), both bound to lamins A/C, activates c-Fos/AP-1-driven transcription (Gonzalez et al. 2008). Furthermore, ERK-mediated displacement of retinoblastoma protein (pRb) from lamins A/C promotes its cdk-dependent phosphorylation (Rodriguez et al. 2010). Lamin scaffolds can also recruit nuclear phosphatase PP2A, promoting dephosphorylation of pRb (Van Berlo et al. 2005). In addition, lamins also indirectly affect signaling via the binding and regulation of integral proteins of the INM, such as emerin and MAN1 (Vaughan et al. 2001; Liu et al. 2003), which affect signaling pathways. MAN1 binds to and deactivates Smads, components of the transforming growth factor β (TGFβ) pathway (Lin et al. 2005), and emerin mediates nuclear export of β-catenin, a downstream factor of Wnt signaling (Markiewicz et al. 2006; Tilgner et al. 2009).

Interestingly, emerin has also been implicated in mechanosignaling. It was shown to exert capping activity toward pointed ends of actin filaments, thereby increasing actin polymerization (Holaska et al. 2004), which in turn regulates the mechanosensitive transcription factor megakaryoblastic leukemia 1 (MKL1), particularly in the cardiovascular system (Ho et al. 2013). MKL1 is a myocardin family member promoting up-regulation of serum response factor (SRF) genes, including actin and vinculin (Miralles et al. 2003). Binding of MKL1 to G-actin inhibits its nuclear import and promotes its nuclear export, thereby attenuating the SRF response (Mouilleron et al. 2008).

Besides lamins residing in the nuclear lamina, the nucleoplasmic A-type lamins (Dechat et al. 2010b) together with their binding partner, LAP2α (Dechat et al. 2000), have also been implicated in signaling and gene expression. The lamin A/C–LAP2α complex has been shown to regulate pRb/E2F signaling (Markiewicz et al. 2002; Dorner et al. 2006; Pekovic et al. 2007) and thereby serves as key determinant of cell fate, keeping the well-balanced state between proliferation and differentiation of tissue progenitor cells (Gesson et al. 2014). Loss of LAP2α shifts this balance toward increased proliferation, potentiating the stemness character (Naetar et al. 2008; Gotic et al. 2010). Depletion of LAP2α in mice causes selective loss of A-type lamins in the nucleoplasm, while Lmna-deficient mouse muscle cells up-regulate LAP2α levels (Naetar et al. 2008; Cohen et al. 2013). Although our knowledge about the regulation and functions of nucleoplasmic lamins A/C is still very sparse, it adds a completely new perspective to our current view on lamin A/C-mediated signaling.

Finally, aside from directly regulating components of cell signaling pathways, lamin complexes can also affect gene expression by organizing higher-order chromatin structure and regulating epigenetic pathways. Complexes of A-type lamins and the lamin B receptor (LBR), an integral INM protein, were shown to redundantly anchor heterochromatin to the lamina (Solovei et al. 2013). In addition, ablation of the single lamin gene in C. elegans or expression of a mutated muscle dystrophy-linked lamin was found to impair the tissue-specific regulation of the heterochromatin/lamina interaction during C. elegans development (Mattout et al. 2011). Genomic regions associated with the lamina, called lamina-associated domains (LADs), are up to 10 Mb long, enriched in heterochromatic epigenetic marks, gene-poor, and transcriptionally inactive (Guelen et al. 2008; Peric-Hupkes et al. 2010). It has been proposed that lamina anchoring contributes to the stable silencing of genes during differentiation in a cell type-specific manner (Shevelyov et al. 2009; Meister et al. 2010; Towbin et al. 2010; Kohwi et al. 2013). A recent study showed that binding of lamin A to subregions in promoters influences gene expression dependent on the presence and localization of active and repressive histone marks within the promoter (Lund et al. 2013). However, the molecular mechanism underlying the regulation of gene expression by tethering chromatin to the lamina is still unclear. Interestingly, the binding of lamin A to promoters does not appear to be restricted to the nuclear periphery but occurs throughout the nuclear interior, involving nucleoplasmic lamin A complexes (Lund et al. 2013).

The route and components of mechanosensing

Mechanosensing defines the ability of cells to react to external forces such as stretch, shear stress, and compression, enabling them to sense and adapt to mechanical changes in their environment. Primary mechanosensors are located at the plasma membrane and can directly mediate the conversion of mechanical signals into biochemical signals (Fig. 1). Such sensors comprise integrins mediating cell attachment to the ECM, cell–cell adhesion molecules such as platelet endothelial cell adhesion molecule-1 (PECAM1), vascular endothelial growth factor receptor 2 (VEGFR2), mechanosensory complexes on endothelial cells such as VE cadherin (Fig. 2), and stretch-activatable ion channels (Tzima et al. 2005; Davies 2009; Wolfenson et al. 2013; Janostiak et al. 2014; Leckband and de Rooij 2014; Yao et al. 2014). Mechanostimulation usually results in stretching of the primary mechanosensor itself, unmasking cryptic binding sites for various molecules. For instance, stretching of integrins results in their conversion to a “high-affinity state,” increasing the recruitment of focal adhesion (FA) components such as vinculin and activating a signaling cascade involving FA kinase (FAK), Src, and ERK (Schwartz 2009). Prolonged mechanostimulation leads to long-term changes in gene expression, such as the up-regulation of endothelial nitrite oxide synthase (eNOS) in endothelial cells upon shear stress (Davis et al. 2004).

Figure 1.

The route of mechanosensing and the tension-induced reinforcement response. (Top panels) Cell–cell adhesion and FA complexes that sense tension are physically linked to the nucleus via the cytoskeleton, LINC complexes (SUN and KASH domain proteins) in the nuclear membrane, and the nuclear lamina. Tension forces from the ECM are transmitted into the nucleus via these components and affect mechanoresponsive gene expression. (Bottom panels) In response to a mechanostimulus, such as increase in ECM stiffness, adhesion complexes, the actin cytoskeleton, LINC complexes, and the lamina are reinforced by the assembly of actin filaments, increased recruitment of adhesion complex and LINC complex proteins, and stabilization and assembly of A-type lamins at the lamina, thereby counteracting forces exerted from outside. In addition, the INM protein emerin becomes phosphorylated and contributes to LINC complex reinforcement. Tension also induces the activation of signaling cascades on adhesion complexes, such as FAK signaling, which affects mechanoresponsive gene expression without direct force transmission into the nucleus. Panels at the right depict higher-magnification views of the boxed areas in the nucleus shown in the left panels.

Figure 2.

Shear stress force induces rearrangement of the cytoskeleton and cell alignment. Lung endothelial cells were exposed to flow shear stress (12 dyn/cm^{) for 3 h and processed for immunofluorescence microscopy using actin and VE-cadherin antibodies. Hoechst was used for DNA staining. Shear stress induces increased alignment of actin stress fibers and elongation of nuclei (arrows) in the flow direction. Note “reinforcement” of cell–cell junctions particularly at the posterior and anterior ends of polarized cells as revealed by increased accumulation and “zipper-like” morphology of VE-cadherin junctions and increased cortical actin at these sites (arrowheads). Bar, 20 μm.}

Many studies have emphasized the necessity of an intact cytoskeleton for the mechanoactivation of signaling molecules such as FAK and NFκB (Wang et al. 2009b). Davies (2009) proposes that efficient transmission of tension signals requires a well-interconnected and intact cytoskeleton. In support of this model, several studies have shown long-range cytoskeleton-mediated force propagation within the cytoplasm that can also be directly transmitted to the nucleus (Fig. 1; Maniotis et al. 1997; Hu et al. 2005; Wang et al. 2005). Similarly, the “hardwired” model proposes direct transmission of forces to the nucleus via integrins (Wang et al. 2009a). Consistent with this model, it has recently been shown by monitoring movements of fluorescently tagged nucleolar marker proteins that extracellular forces, such as compression and shear stress, can stimulate movements inside the nucleus, which, after prolonged exposure, are directly correlated in magnitude and direction to the applied stress (Booth-Gauthier et al. 2012). Thus, altering the mechanical environment of the cell can ultimately lead to long-term changes in nuclear organization and gene expression patterns. Altogether, activation of mechanoresponsive genes can be achieved via two main routes: one involving stretch-induced cell adhesion complex- and cytoskeleton-mediated activation of signaling molecules and transcriptional regulators at the plasma membrane and a second involving a direct force transmission into the nucleus (Fig. 1).

Direct force transmission into the nucleus requires intact physical connections of the nucleus to the cytoskeleton (Fig. 1). Such connections are mediated by the members of the so-called linker of nucleoskeleton and cytoskeleton (LINC) complex (Crisp et al. 2006; Tapley and Starr 2013). LINC complexes comprise SUN domain proteins that span the INM and are in the perinuclear space physically connected to nesprins, KASH domain proteins residing in the outer nuclear membrane (Sosa et al. 2012). Different types of nesprins either bind actin directly or interact via microtubule motor proteins and plectin with microtubules and IFs, respectively (Mejat and Misteli 2010). LINC components are crucial determinants of nuclear morphology, as nuclear height was shown to increase upon depletion of nesprin-1 in endothelial cells, presumably due to the lack of tensional forces exerted on the nucleus by actin cables (Chancellor et al. 2010). In support of their role in the direct transmission of forces to the nucleus, the nuclear deformation response to mechanical strain was found abrogated upon LINC disruption using dominant-negative SUN and nesprin constructs (Lombardi et al. 2011). Short-term activation of mechanosensitive genes, however, appeared unchanged in these LINC-disrupted cells. In contrast, activation of the wound healing response that requires activation of the mechanoresponsive signaling machinery was impaired in cells deficient for nesprin-2 (Rashmi et al. 2012), suggesting impaired mechanotransduction in these cells. Thus, it is still unclear to what extent LINC family members are essential for activation of mechanotransduction pathways.

LINC family members concentrate at specialized apical regions of the nucleus, the so-called transmembrane actin-associated nuclear (TAN) lines (Luxton et al. 2010). Sun-2 and Nesprin-2 giant are essential components of these TAN lines, which associate with dorsal actin cables at the cytoplasmic surface of the nucleus and are anchored via lamins to the nuclear interior. The INM proteins Samp and emerin (Borrego-Pinto et al. 2012; Chang et al. 2013) were also found in these complexes. TAN lines at the nuclear membrane resemble in many aspects FA complexes at the plasma membrane. Both are linked to actin fibers, and their formation is enhanced in response to increased contractility of the system upon serum stimulation or mechanostimulation, such as exposure to shear stress (Chambliss et al. 2013). Depletion of LINC complex components results in the inability of cells to respond to a shear stimulus and in defective cell polarization and migration (Luxton et al. 2010; Chambliss et al. 2013). Thus, TAN lines in analogy to FA sites represent major nuclear mechanosensing structures that are likely also involved in nuclear mechanotransduction (see below for possible mechanisms).

The most downstream components of the mechanosensing pathway from the ECM into the nucleus are the major nuclear mechanosensors, the A-type lamins (Fig. 1). It is their scaffolding property, as mentioned above, and their strategic position at the interface between LINC complexes in the nuclear membrane and chromatin that position them as key molecules of the nucleocytoskeletal coupling machinery. This notion is supported by studies showing that cells lacking A-type lamins or expressing lamin A mutants are unable to directly transmit forces to the nucleus (Poh et al. 2012; Zwerger et al. 2013) and that nuclei of these cells do not deform upon mechanostimuli (isotropic response) (Houben et al. 2007). In addition, the localization of various components of the LINC complex is severely disturbed in these cells (Hale et al. 2008; Chen et al. 2012, 2014), and the connection of the nuclei to TAN lines is weakened or lost (Folker et al. 2011). Importantly, this defective nucleocytoskeletal coupling appears to ultimately lead to defects in mechanotransduction, as reflected by the impaired activation of NFκB (Lammerding et al. 2004) and Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) (Swift et al. 2013; Bertrand et al. 2014) and MKL/SRF signaling (Ho et al. 2013).

Molecular mechanisms of lamins in mechanosensing/signaling

‘Outside-in signaling’

The intriguing question arising at this point is how lamins propagate tensional forces to the nucleus. Recent data from the Disher laboratory (Swift et al. 2013) studying structural changes in lamin A upon mechanostimulation shed some light on this question (Fig. 3). Exposure of nuclei to shear stress induced stretching of lamin A filaments due to partial “unfolding” of the lamin Ig-fold. Interestingly, the Ig-fold in lamin A was recently shown to define the viscoelastic properties of lamin A and the mechanical resilience of lamin A networks (Bera et al. 2014).

Figure 3.

Lamin A in mechanosignaling. Increasing tension causes partial unfolding and dephosphorylation of lamin A as well as assembly of soluble lamin A into the lamina. These structural and biochemical changes of the protein may affect chromatin organization and cell signaling, thereby activating the reinforcement response, including increased lamin A expression and remodeling of the cytoskeleton and the ECM.

Additional evidence for a structural and/or biochemical response of lamin A proteins to mechanical stimuli is demonstrated by dephosphorylation of lamin A at Ser22 and Ser390 upon increased matrix stiffness and cell tension (Buxboim et al. 2014). Lamin phosphorylation at these sites is required for mitotic lamina disassembly (Heald and McKeon 1990) and promotes dissociation of lamin A from the lamina and distribution throughout the nucleus in interphase cells (Kochin et al. 2014). Therefore, it was proposed that stress-mediated changes in lamin A/C phosphorylation at these sites may regulate lamin A assembly into the lamina and its stability (Buxboim et al. 2014). In this model, increased tension would favor dephosphorylation of lamins, leading to its assembly into the lamina and an increase in protein stability (Fig. 3). How mechanical tension affects lamin A phosphorylation is still unclear, but it is conceivable that the degree of lamin stretching may affect phosphorylation and/or dephosphorylation by changing the access for kinases or phosphatases. In line with this, Swift et al. (2013) have shown that a lamin A protein with a muscular dystrophy-causing alteration in the Ig-fold (R453W) is less phosphorylated and that this is correlated with an apparently higher degree of protein unfolding. It must be emphasized though that our knowledge of tensional changes in lamins and consequences of such alterations for the protein and the cell is still sparse and requires further elucidation.

Regardless of the exact mode of action, “stretched lamins” could further relay the tensional input to the nuclear interior by the following not mutually exclusive mechanisms (Fig. 3; Isermann and Lammerding 2013): Changes in the mechanical properties of A-type lamins and the lamina may (1) alter chromatin organization and accessibility for chromatin regulators, (2) cause chromatin detachment from the transcriptionally repressive periphery, and (3) alter the interaction of signaling molecules with A-type lamins. This model is particularly intriguing in view of the emerging concept that both A-type lamins associated with the peripheral lamina and A-type lamins in the nuclear interior affect chromatin organization, signaling, and gene expression (Dechat et al. 2010b). Thus, changes in the ratio of lamina-associated versus nucleoplasmic pools of lamins A/C may significantly affect the cellular phenotype.

Recent data on isolated nuclei have shown that lamin A also mediates the tension-mediated nuclear stiffening response (Fig. 1). Using magnetic tweezers to apply forces on nesprins in isolated nuclei resulted in higher nuclear stiffness through increased recruitment of lamins to LINC complexes (Guilluy et al. 2014). These forces also induced emerin phosphorylation, which appears to act as tension sensor. In the absence of lamins, nuclei exhibit a reduced resistance to forces and cannot respond to tension by nuclear stiffening. In line with such a mechanism, nuclei lacking functional lamin A/C or emerin are unable to resist forces exerted by TAN lines despite unperturbed assembly of Sun and Nesprin-2 giant (Folker et al. 2011; Chang et al. 2013). In these cells, TAN lines were found to slip over nuclei rather than move with them.

Such tension-mediated reinforcement mechanisms were long known to operate at the “cytoplasmic” level. ECM-exerted forces on integrins induce assembly and phosphorylation of FA components and increased actin-driven contractility that in turn promote ECM assembly in a feedback loop (Fig. 2; Galbraith et al. 2002; Icard-Arcizet et al. 2008; Schwartz 2009; Humphrey et al. 2014). It still awaits to be seen whether, similar to FAs, “nuclear adhesion” also involves a plethora of other components besides emerin and whether these can directly signal to actin filaments. This is supported by the observation that cells expressing emerin phosphorylation mutants exhibited a significant reduction in actin stress fibers (Guilluy et al. 2014).

‘Outside-in signaling’

The intriguing question arising at this point is how lamins propagate tensional forces to the nucleus. Recent data from the Disher laboratory (Swift et al. 2013) studying structural changes in lamin A upon mechanostimulation shed some light on this question (Fig. 3). Exposure of nuclei to shear stress induced stretching of lamin A filaments due to partial “unfolding” of the lamin Ig-fold. Interestingly, the Ig-fold in lamin A was recently shown to define the viscoelastic properties of lamin A and the mechanical resilience of lamin A networks (Bera et al. 2014).

Figure 3.

Lamin A in mechanosignaling. Increasing tension causes partial unfolding and dephosphorylation of lamin A as well as assembly of soluble lamin A into the lamina. These structural and biochemical changes of the protein may affect chromatin organization and cell signaling, thereby activating the reinforcement response, including increased lamin A expression and remodeling of the cytoskeleton and the ECM.

Additional evidence for a structural and/or biochemical response of lamin A proteins to mechanical stimuli is demonstrated by dephosphorylation of lamin A at Ser22 and Ser390 upon increased matrix stiffness and cell tension (Buxboim et al. 2014). Lamin phosphorylation at these sites is required for mitotic lamina disassembly (Heald and McKeon 1990) and promotes dissociation of lamin A from the lamina and distribution throughout the nucleus in interphase cells (Kochin et al. 2014). Therefore, it was proposed that stress-mediated changes in lamin A/C phosphorylation at these sites may regulate lamin A assembly into the lamina and its stability (Buxboim et al. 2014). In this model, increased tension would favor dephosphorylation of lamins, leading to its assembly into the lamina and an increase in protein stability (Fig. 3). How mechanical tension affects lamin A phosphorylation is still unclear, but it is conceivable that the degree of lamin stretching may affect phosphorylation and/or dephosphorylation by changing the access for kinases or phosphatases. In line with this, Swift et al. (2013) have shown that a lamin A protein with a muscular dystrophy-causing alteration in the Ig-fold (R453W) is less phosphorylated and that this is correlated with an apparently higher degree of protein unfolding. It must be emphasized though that our knowledge of tensional changes in lamins and consequences of such alterations for the protein and the cell is still sparse and requires further elucidation.

Regardless of the exact mode of action, “stretched lamins” could further relay the tensional input to the nuclear interior by the following not mutually exclusive mechanisms (Fig. 3; Isermann and Lammerding 2013): Changes in the mechanical properties of A-type lamins and the lamina may (1) alter chromatin organization and accessibility for chromatin regulators, (2) cause chromatin detachment from the transcriptionally repressive periphery, and (3) alter the interaction of signaling molecules with A-type lamins. This model is particularly intriguing in view of the emerging concept that both A-type lamins associated with the peripheral lamina and A-type lamins in the nuclear interior affect chromatin organization, signaling, and gene expression (Dechat et al. 2010b). Thus, changes in the ratio of lamina-associated versus nucleoplasmic pools of lamins A/C may significantly affect the cellular phenotype.

Recent data on isolated nuclei have shown that lamin A also mediates the tension-mediated nuclear stiffening response (Fig. 1). Using magnetic tweezers to apply forces on nesprins in isolated nuclei resulted in higher nuclear stiffness through increased recruitment of lamins to LINC complexes (Guilluy et al. 2014). These forces also induced emerin phosphorylation, which appears to act as tension sensor. In the absence of lamins, nuclei exhibit a reduced resistance to forces and cannot respond to tension by nuclear stiffening. In line with such a mechanism, nuclei lacking functional lamin A/C or emerin are unable to resist forces exerted by TAN lines despite unperturbed assembly of Sun and Nesprin-2 giant (Folker et al. 2011; Chang et al. 2013). In these cells, TAN lines were found to slip over nuclei rather than move with them.

Such tension-mediated reinforcement mechanisms were long known to operate at the “cytoplasmic” level. ECM-exerted forces on integrins induce assembly and phosphorylation of FA components and increased actin-driven contractility that in turn promote ECM assembly in a feedback loop (Fig. 2; Galbraith et al. 2002; Icard-Arcizet et al. 2008; Schwartz 2009; Humphrey et al. 2014). It still awaits to be seen whether, similar to FAs, “nuclear adhesion” also involves a plethora of other components besides emerin and whether these can directly signal to actin filaments. This is supported by the observation that cells expressing emerin phosphorylation mutants exhibited a significant reduction in actin stress fibers (Guilluy et al. 2014).

‘Inside-out’ signaling

Recent findings from the Discher laboratory (Swift et al. 2013; Buxboim et al. 2014) show that cells not only adjust to increased forces by reinforcing the coupling of lamins to LINC complexes but also increase lamin A protein levels in response to force. Tension exerted through myosin-mediated actin stress fiber assembly following plating of cells on a stiff matrix promotes dephosphorylation and stabilization of the lamin A protein within 30 min. Long-term responses and adjustment to increased matrix stiffness, as occurs during osteogenic differentiation, require several days and involve increased expression of the lamin A gene. Increased tension also leads to the up-regulation of the mechanosensitive SRF pathway that in turn up-regulates myosin IIA. This so called “mechanobiological gene circuit” is a feedback loop to increase tension in the cell. It requires intact nucleocytoskeletal mechanocoupling, as disruption of the LINC complex and the actin cytoskeleton perturbs such a circuit.

Very little is known about the exciting features of lamin A-mediated inside-out signaling, but a few potential mechanisms are emerging: A-type lamins are required for the correct localization of emerin in the INM (Sullivan et al. 1999), which regulates actin assembly (Holaska et al. 2004) and the mechanosensitive MKL1–SRF pathway (Ho et al. 2013). In addition, emerin is known to affect wnt signaling (Markiewicz et al. 2006), which in turn regulates expression of ECM components (Hernandez et al. 2010). In line with this model, a growing amount of data shows that mutations in A-type lamins affect the composition of the ECM. Lamin A/C-deficient fibroblasts exhibited increased collagen synthesis, and, importantly, re-expression of lamins A/C reverted this phenotype (Van Berlo et al. 2005). In addition, HGPS patient cells expressing progerin and ZMPSTE24-deficient mice expressing prelamin A (Hernandez et al. 2010; de la Rosa et al. 2013) showed aberrant ECM production. In particular, proteoglycans (Beavan et al. 1993), glycoproteins, and collagen XI that regulate collagen assembly (Hernandez et al. 2010) and many other ECM components (Csoka et al. 2004) were affected. Interestingly, these changes were linked to altered TGF-β signaling in Lmna ^{cells and wnt/β-catenin signaling in HGPS cells (}Van Berlo et al. 2005; Hernandez et al. 2010). While it is well known that Rho-activated cell contractility and tension modulate the expression of ECM components in a TGF-β-dependent manner (Chapados et al. 2006; Marenzana et al. 2006; Meyer-ter-Vehn et al. 2006), the role of A-type lamins in this “mechanobiological gene circuit” is still unclear (Buxboim et al. 2014).

Another potential mechanism as to how tension-induced changes in lamin A protein levels and localization may affect gene expression comes from recent studies showing that binding of lamin A to promoters may regulate gene expression during adipocyte differentiation (Lund et al. 2013).

Lamin A in cell migration

Activation of Rho-GTPases is one of the primary cellular responses to mechanical stimuli that leads to extensive cytoskeletal rearrangement, cell polarization, and migration (Houben et al. 2007). During wound healing-induced migration, cytoskeletal remodeling is accompanied by increased attachment of actin stress fibers to nuclear TAN lines and dorsal movements of actin cables dragging nuclei away from the wound edge. At the same time, repositioning of the centrosome toward the leading edge occurs. A-type lamins appear essential for these processes, since, in their absence, cells fail to reposition their nuclei, and centrosomes fail to polarize (Luxton et al. 2010; Folker et al. 2011). However, nuclear repositioning does not appear crucial for the overall two-dimensional (2D) cell migration, since lamin A deficiency results only in a mild reduction in migration speed (Hale et al. 2008). In contrast, migration through a constrictive micrometer-scale pore three-dimensional (3D) environment is significantly delayed in cancer cells or neutrophils with high lamin A levels. This is conceivable since A-type lamins affect nuclear morphology and its visco-elastic properties, increasing nuclear stiffness (see above) and thus allowing only limited compressibility. Consistent with this notion, HGPS cells with increased nuclear stiffness (Dahl et al. 2006) show slowed down migration through 6-μm-thick constrictions (Booth-Gauthier et al. 2013), whereas lamin A knockdown increased the migration speed through constrictions but reduced overall resistance of cells to stress (Harada et al. 2014).

Role of lamin-mediated mechanosensing in disease

Mutations in the LMNA gene cause ∼14 different diseases termed laminopathies (Worman 2012). The majority of disease-causing mutations in LMNA are autosomal dominant missense mutations, leading to a single amino acid change in lamins A and C. Depending on the localization in the gene, these mutations can interfere with protein folding (Bollati et al. 2012), stability, and assembly (Wiesel et al. 2008; Bank et al. 2012) or change the biochemical properties of the protein (Krimm et al. 2002). Most of the HGPS cases are caused by a silent mutation in LMNA, leading to missplicing of the LMNA transcript and production of a slightly smaller form of lamin A that, unlike wild-type lamin A, maintains its farnesylated and carboxymethylated C-terminal cysteine (Worman 2012).

In the context of mechanosensing, mutations affecting mechanical load-bearing tissues are of particular interest, such as striated muscle in EDMD and DCM and smooth muscle and cartilage/bone in accelerating aging disorders. Interestingly, similar disease phenotypes like in LMNA-linked EDMD and DCM can also be observed by mutations in EMD (encoding emerin) and in genes encoding LINC complex family members, such as SYNE1 and SYNE2 (encoding nesprin-1 and nesprin-2) and SUN1 and SUN2 (encoding SUNs) (Schreiber and Kennedy 2013; Meinke et al. 2014). These data strongly suggest that defective nucleocytoskeletal coupling is a major cause for the disease phenotypes in muscle. In line with this model, cells harboring these myopathic lamin mutations had impaired nuclear stability and defective nuclear force transmission (Folker et al. 2011; Zwerger et al. 2013). Myopathic lamin mutations but not those causing phenotypes in adipose tissues also caused defective TAN line anchoring (Folker et al. 2011). Furthermore, lamin A/C-deficient mice, characterized by severe DCM, show absence of desmin attachments to the nucleus in cardiomyocytes and did not develop a “hypertrophic mechanoresponse” (Nikolova et al. 2004).

Similarly, HGPS-linked defects in mechanical load-bearing tissues, such as smooth muscle and bone, may be caused by aberrant nucleocytoskeletal coupling. In accordance with this, accumulation of progerin was shown to cause disturbances in LINC complexes. For instance, farnesylated lamin (prelamin and progerin) showed increased affinity for SUN proteins, thereby reducing their mobility (Chen et al. 2014). Interestingly, deletion of SUN1 in both Lmna ^{mice and a progeria mouse model ameliorated the disease phenotypes (}Chen et al. 2012; Suh and Kennedy 2012). Progerin/prelamin also showed increased binding to emerin, and prelamin expression strongly influenced the localization of emerin (Capanni et al. 2009; Wu et al. 2014).

As mentioned above, one of the possible consequences of disturbed mechanocoupling is the altered expression of ECM components. This is particularly relevant in view of the fact that the hallmark of many laminopathies is increased fibrosis (increased collagen production) (Reichel and Garcia-Bunuel 1970; Van Berlo et al. 2005; Worman 2012). DCM is very often associated with increased expression of Tgfb1/Tgfb2 and Col1a genes (Margulies et al. 2009). Furthermore, cardiovascular pathology in HGPS was characterized by very pronounced collagen accumulation, which leads to severe atherosclerosis, myocardial infarction, and stroke at a very early age (Olive et al. 2010). Thus, it is conceivable that the accelerated aging process in HGPS is partially caused by dysfunctional ECM production. The aging process itself has been associated with defective ECM production. For example, the reduced self-renewal and bone formation capacity of aged mesenchymal stem cells (MSCs) was corrected by culturing them on ECM from young MSCs (Sun et al. 2011). In analogy to that, proliferative defects of mouse adult fibroblasts harboring HGPS-linked Lmna mutation were rescued upon growth on ECM derived from wild-type cells (Hernandez et al. 2010). Similar observations were reported for Zmpste24 ^{cells (}de la Rosa et al. 2013). This study also showed that, in contrast to the severely reduced life span in mice completely lacking ZMPSTE24, the ZMPSTE24 mosaic mice containing equal numbers of ZMPSTE-deficient (prelamin A-accumulating) and ZMPSTE-proficient (mature Lamin A-containing) cells in the tissues develop normally. Altogether a “well-balanced relationship” between the nuclear lamina and the extracellular environment may be a key factor toward a delayed aging response.