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Nesprins LINC the nucleus and cytoskeleton
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Nesprins LINC the nucleus and cytoskeleton Jason A Mellada, Derek T Warrena and Catherine M Shanahan Like other spectrin repeat proteins, nesprins co-ordinate and maintain cellular architecture by linking membranous organelles to the cytoskeleton. However nuclear envelope (NE) nesprins, uniquely hardwire the nuclear lamina to the cytoskeleton and molecular motors. Emerging evidence suggests that nesprins also form a continuous network linking the plasma membrane to the NE that potentially translates mechanical stimuli into nuclear reorganisation. Surprisingly, this network is also essential for cytoskeletal organisation and its disruption has dramatic effects on nuclear migration, centrosomal positioning, focal adhesion maturation and cell motility. Herein we review recent advances in our understanding of how nesprins couple to various filamentous systems within the cell and emphasise the importance of both KASH and KASH-less nesprin isoforms in these interactions. Address BHF Centre of Research Excellence, Division of Cardiovascular Medicine, King’s College London, SE5 9NU, UK a These authors contributed equally. Corresponding author: Shanahan, Catherine M ([email protected])
Current Opinion in Cell Biology 2011, 23:47–54 This review comes from a themed issue on Cell structure and dynamics Edited by Anne Ridley and Rebecca Heald Available online 20th December 2010 0955-0674/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2010.11.006
Introduction The nuclear envelope (NE) is a highly organised structure that partitions the nuclear and cytoplasmic environments of the cell. It is composed of two lipid bilayers, the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) and these are separated by a lumenal space [1,2]. Nuclear pore complexes span these membranes and regulate protein transport between the nucleoplasm and the cytosol [1,2]. NE structural and functional integrity is maintained via specific components of the INM and ONM that also physically link the cytoskeleton and nuclear lamina; a meshwork of intermediate filaments (IF) consisting of A-type and B-type lamins that underlie the INM (Figure 1) [3]. These essential connections enable the nucleus to maintain its position and tension within the cell during cell www.sciencedirect.com
polarisation, migration and differentiation and also relay mechanical signals to the nucleus [4]. The evolutionarily conserved, multi-isomeric Nuclear Envelope SPectRIN repeat proteins (nesprins) are an emerging family of nuclear-cytoplasmic couplers. Four genes (SYNE 1–4) encoding nesprins 1–4 have been identified so far. Nesprins are characterised by a central rod region of variable length, comprised of multiple spectrin repeats, and a C-terminal Klarsicht/ANC-1/Syne homology (KASH) transmembrane domain that facilitates NE localisation [5]. The nesprins are also characterised by variable N-terminal motifs that enable interactions with different components of the cytoskeleton. Giant isoforms of nesprin1 (1 MDa in size) and nesprin-2 (800 kDa) have Nterminal calponin homology (CH) domains that link to filamentous actin (F-actin), nesprin-3 (110 kDa) has a plectin-binding motif that permits interactions with cytoplasmic IFs while nesprin-4 interacts indirectly with microtubules [5–7]. Importantly, nesprins localise via the KASH domain to both the INM and ONM and are partitioned to these domains depending on their size and binding partners (Figure 1) [8]. INM nesprins-1/2 possess C-terminal spectrin repeats that can interact with both the INM protein emerin and A-type lamins [9,10]. Thus, nesprins can form structural connections on either face of the NE. Nesprins and other KASH-domain proteins are recruited to the NE by binding to the Sad1p-UNC84 (SUN) domain proteins Sun1 and Sun2 within the perinuclear space, forming the linker of the nucleoskeleton and cytoskeleton (LINC) complex (Figure 1) [8]. The LINC anchors the nuclear lamina, which binds INM Sun proteins, to the cytoskeleton via ONM nesprins. Overexpression of dominant-negative KASH domain constructs and knockdown of LINC components nesprins-1/2, Sun1/2 or lamin A uncouples the INM from the ONM, detaches the nucleus from the cytoskeleton and decreases mechanical stiffness [8]. The importance of the LINC in cytoskeletal organisation and biomechanical signalling is further reflected in the wide range of disease phenotypes resulting from mutations in nesprins-1/2, including cardiac and skeletal muscle defects in Emery Dreifuss muscular dystrophy (EDMD), cerebellar defects in autosomal recessive spinocerebellar ataxia 1 (ARCA1) and tendon contractures in autosomal recessive arthrogryposis multiplex congenita (AMC) [11–13].
Nesprins associate with multiple cytoskeletal systems The LINC has primarily been characterised as serving three functions: to tether the nucleus to the cytoskeleton, Current Opinion in Cell Biology 2011, 23:47–54
48 Cell structure and dynamics
Figure 1
Actin cytoskeleton Intermediate filaments
Microtubules Nesprin-4
Nesprin-3
Cytoplasm
Nesprins-1/2
Plectin
Kif5B
ONM Nuclear pore complex
SUN domain
SUN domain
SUN domain
SUN domain INM
Emerin Nucleoplasm
CH domain
Nuclear lamina
Spectrin repeat
KASH domain
SUN domain containing protein Current Opinion in Cell Biology
The LINC complex facilitates the coupling of the nuclear lamina to cytoplasmic cytoskeletal systems via the nesprins. The LINC complex is comprised of SUN domain containing proteins (SUN1 and 2, UNC84 and SPAG4) on the INM that bind to the nuclear lamina via interactions with lamin A, and potentially smaller nesprin-1/2 isoforms and emerin. The KASH domain of the larger isoforms of nesprins-1/2, nesprin-3 and nesprin-4 at the ONM associate with the SUN domain within the luminal space to tether the NE to either cytoplasmic actin, IFs or microtubule networks.
facilitate the mechanotransduction of peripheral signals to the nucleus and also to regulate cytoplasmic filament organisation during migration, adhesion and polarity establishment. It is clear that ONM nesprins are integral to all of these roles owing to their ability to indirectly or directly interact with all three major cytoplasmic filaments (Table 1). Connections with the actin cytoskeleton
The most characterised nesprin LINCs anchor the nucleus to the actin cytoskeleton. Surprisingly, the direct linkage of the NE to F-actin defines the mechanical properties of the entire cell via mechanotransduction between the cytoplasm and the nucleus [4]. Uncoupling of this mechanical link, in either nesprin-1 KASH domain or C-terminal spectrin repeat knockout mice, results in a loss of cellular tension that induces abnormal localisation of skeletal muscle nuclei and defects in strain transCurrent Opinion in Cell Biology 2011, 23:47–54
mission between the cell and nucleus [14,15]. Moreover, nuclear morphology is defined by thick actin bundles that are organised into a curved shell overlaying the nucleus, moulding it into a shape resembling overall cell morphology. Chemicals that depolymerise F-actin or inhibit Rho kinase, a key regulator of F-actin organisation and actomyosin contractility, release the nucleus from these physical constraints, resulting in nuclear morphology defects [16]. Similarly, disruption of the LINC complex, by either the expression of dominantnegative nesprin KASH constructs or mutation of nuclear lamina proteins, eliminates this actin shell and induces nuclear morphology defects [16]. Furthermore, uncoupling of the nucleus from the actin cytoskeleton triggers a loss of cellular tension. This loss of tension impacts on maturation of focal adhesions, important macromolecular cell–matrix adhesions that transmit mechanical force and other signals, as well as on cell motility. This suggests that www.sciencedirect.com
Nesprins LINC the nucleus and cytoskeleton Mellad, Warren and Shanahan 49
Table 1 Summary of demonstrated nesprin LINC complexes. Nesprins connect the NE to the cytoskeleton via direct interactions with F-actin or indirectly to microtubule and IF binding proteins. Connection Actin
F-actin
Nesprin
Function
Meckelin
Nesprin-1 Nesprin-2 Nesprin-2
IF
Lamin A Plectin
Nesprin-1/2 Nesprin-3
Chromatin organisation, NE architecture. NE-IF coupling
Microtubules
Kinesin-1 Kinesin-2 Dynein Dynactin
Nesprin-2/4 Nesprin-1 Nesprin-1/2 Nesprin-1/2
Nuclear migration, polarity. Vesicular transport. Nuclear migration, polarity. Nuclear migration, polarity
nuclear tethering to the actin cytoskeleton may be crucial for F-actin organisation throughout the cell [17,18,19]. Although the N-terminal paired CH domains remain the only characterised F-actin-binding module within nesprins-1/2 [20,21], evidence now exists for alternative connections to the actin cytoskeleton. For instance, nesprin-2 CH domain knockout dermal fibroblasts display nuclear blebbing and lobulations in vitro characteristic of LINC disruption. Interestingly, after several passages, these fibroblasts gradually express a novel, N-terminally truncated giant nesprin-2 isoform that alleviates this phenotype [22]. This suggests that similar to their homologues dystrophin and utrophin, nesprin spectrin repeats may also bind filamentous actin either directly or indirectly via as yet unknown binding partners [23–25]. Intriguingly, emerging data support the existence of Factin at the INM. Recent studies have shown that the nesprin binding partners emerin and lamin-A serve to stabilise and bundle F-actin [26,27]. Importantly, Factin may potentially contribute to LINC stability as emerin, lamin A and nesprin-1/2 spectrin repeats can all interact with the nucleoplasmic N-termini of Sun1/2 [28,29,30,31]. However, the nature and abundance of actin filaments at the INM remains controversial and further work is required to determine if INM nesprins and nuclear actin are also components of the LINC complex.
Connections with the centrosome/ microtubule network Nesprins also make multiple indirect connections with the microtubule network by forming microtubule motor LINCs. Analogous to the roles of UNC-83 and ZYG-12 in C. elegans and Klarsicht in Drosophila, these LINCs are essential for both nuclear and centrosomal positioning and therefore cell polarity [8]. Recent studies have demonstrated that nesprins-1/2 interact with dynein/ dynactin and kinesin-1 in the developing mouse brain and loss of these interactions in double nesprin-1/2KASH knockout mice uncouples the centrosome from www.sciencedirect.com
Nuclear positioning, Mechanotransduction. Ciliogenesis.
the neuronal nucleus resulting in severe nucleokinesis and interkinetic nuclear migration defects [30]. Similarly, zebrafish nesprin-2 also binds to dynactin and is necessary for photoreceptor interkinetic nuclear migration as demonstrated by knockdown and dominant-negative KASH overexpression studies [32]. Moreover, interactions between nesprin-4 and kinesin-1 regulate epithelial cell polarity and nuclear positioning with nesprin-4 overexpression dramatically displacing the centrosome and Golgi away from the NE [6]. Interestingly, a central spectrin repeat region of nesprin-1 also binds to the kinesin-2 subunit Kif3b and appears to play a role in cytokinesis and membrane transport. However whether these functions are mediated by a nesprin NE LINC or a KASH-less isoform (see below) must be explored further [33].
Nesprins associate with intermediate filament systems in the nucleus and cytoplasm Nesprins tether to IF systems in both the nucleus and cytoplasm and these connections potentially serve important organisational roles within the cell. The nuclear lamina is an IF meshwork and lamins-A/C were the first binding partners identified for nesprin-1 and nesprin-2. Our understanding of these complexes remains poor and it is not yet clear if these interactions are all LINC-dependent [5]. However both nesprins and lamins bind to multiple integral membrane proteins of the INM and one important function for these nuclear lamina complexes is the organisation of heterochromatin, which is facilitated by auxiliary proteins such as BAF, LAP2a and possibly smaller nesprin-1/2 isoforms [34,35]. Indeed, the dramatic heterochromatin reorganisation induced by knockdown of nesprins-1/2 in U2OS cells and fibroblasts may be partially attributed to the loss of INM isoforms and consequently disruption of a direct link between mechanical stimuli and chromatin organisation, although this possibility requires further investigation [12]. An emerging area for investigation is the interaction between nesprins and cytoplasmic IF networks. These LINCs are proposed to connect the NE to another form of Current Opinion in Cell Biology 2011, 23:47–54
50 Cell structure and dynamics
Figure 2
SR1
SR45
Nesprin-1 Giant 1β 1 1β 2 1α1 1α2
Drop1 CPG2
Nesprin-2 SR1
SR18
Giant 2γ 2β 1 2β 2 2α1 2α2
Nesprin-3
3α CH domain 3β
Spectrin repeat Nesprin-4 KASH domain
4 Current Opinion in Cell Biology
Previously published nesprin isoforms. LINC adaptability is increased by the generation of multiple nesprin isoforms of variable length and with different SR domains. The majority of isoforms characterised so far are N-terminal truncations that retain the KASH domain, suggesting that the proximity of cytoskeletal filaments and other nesprin-bound complexes to the NE may have functional significance. In addition, the number of characterised KASH-less isoforms is steadily increasing.
cell–matrix adhesion known as hemidesmosomes, providing further mechanical support for cells. For example, nesprin-3 binds to the CH domain of the IF-crosslinker plectin and thus forms a continuous IF network between the NE and hemidesmosomes [7,36]. Nesprin-3 appears to be central to IF LINC complex function as both plectin and keratins are drawn to the nuclear periphery of keratinocytes overexpressing nesprin3a [7]. It has been proposed that when actin filaments are naturally or artificially depolymerised the nesprin-3-dependent relocalisation of endogenous plectin to the nuclear periphery may facilitate rapid reorganisation of the cytoskeleton [7]. Current Opinion in Cell Biology 2011, 23:47–54
Different LINCs for different tissues The properties of a mechanically coupled, differentiated muscle cell are vastly different to a fibroblast or a polarised cell such as an endothelial cell. Thus, different cell types have unique biomechanical properties that are likely to require customised connections between the nucleus and cytoskeleton. It is likely that this specialisation is accomplished through the tissue-specific expression of nesprin genes and isoforms allowing cells to fine-tune these connections (Figure 2). For example, nesprin-4 is highly expressed only in specialised secretory epithelium where it is thought to be essential for www.sciencedirect.com
Nesprins LINC the nucleus and cytoskeleton Mellad, Warren and Shanahan 51
establishing centrosome positioning and cell polarity. Similarly, nesprin-1 is essential for neuromuscular junction (NMJ) formation and nesprin-2 is highly enriched in photoreceptor nuclei however the functional reasons for this specificity are not yet clear [6,15,30,32,37]. Nesprin isoforms also vary greatly between different tissues [38]. Nesprins 1/2 are especially abundant in muscle and express muscle specific isoforms. Moreover, mutations
in nesprin-1 and nesprin-2 have been reported in the muscle specific disease EDMD but these mutations appear to have little impact on other tissues. There is also a switch from larger to smaller NE nesprin isoforms during human myotube differentiation. This may serve to adapt LINC function enabling cells to change from a dynamic motile phenotype to a rigid, stable contractile phenotype [39]. It will be interesting to determine
Figure 3
Plasma membrane Binding partners? KASH domain containing isoforms?
KASH-less isoforms?
Actin IFs Microtubules
Cytoplasm
ONM Nuclear pore complex
SUN domain
SUN domain
SUN domain INM
Emerin
Nucleoplasm
Nuclear lamina
Actin filaments
CH domain
Spectrin repeat
KASH domain
SUN domain containing protein Current Opinion in Cell Biology
Nesprins form a mechanotransduction network between the plasma membrane and NE via connections with cytoplasmic cytoskeletal systems. Nesprins localise to the plasma membrane; whether or not these isoforms retain the KASH domain and how they are targeted remain unknown. The nesprin bridging network via the cytoskeleton potentially allows cells to maintain tension between the plasma membrane and nucleus that is essential for both organelle and cytoskeletal organisation within a cell [4]. www.sciencedirect.com
Current Opinion in Cell Biology 2011, 23:47–54
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whether LINCs are also able to dynamically respond to mechanical stimuli thus enabling the cell to rapidly adapt to different environmental conditions. The method of nesprin tethering to the NE may also contribute to LINC functional specialisation. Emerging evidence exists for non-canonical LINCs that do not solely rely on Sun1/2 or lamin A. For instance, Sun1/2 redundantly tether nesprin in some cell types but not in others [36,40,41,42] while torsin-1A, an ATPase localised to the perinuclear space, is also a KASH binding protein that regulates nesprin NE localisation [43,44]. LINCs have recently been implicated in highly specialised roles and have been shown to mediate chromosome pairing during meiosis in C. elegans. It will be important to determine if analogous specialised LINCs also operate in vertebrates [45].
Concluding remarks In conclusion, our knowledge of nesprin LINCs and their role in attaching the NE to the cytoskeleton has increased dramatically over the past few years. However, we still lack a complete understanding of the complexity of connections nesprins make with the filamentous cytoskeletal systems and identification of novel binding partners will help to address this deficit. Additionally, increasing evidence implicates the KASH containing nesprins as signalling scaffolds at the NE and potentially the KASH-less isoforms in other subcellular compartments. However, these KASH-less isoforms remain to be fully characterised. Indeed, a comprehensive read out of nesprin isoform profiles, localisations and interactions in different cell types will enable us to better understand both nesprin and LINC complex variability and function.
Acknowledgements Finally, in addition to their mechanical roles in LINC complex function, recent evidence suggests that nesprins may also serve as signalling scaffold proteins at the NE. Nesprin-2 has recently been shown to regulate WNT signalling and binds alpha and beta catenin at the NE, suggesting that in addition to their structural/mechanical roles LINC complexes may also function as signalling platforms [46].
This work was supported by British Heart Foundation (BHF) grants to CMS.
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multi-isomeric protein that binds lamin and emerin at the nuclear envelope and forms a subcellular network in skeletal muscle. J Cell Sci 2005, 118:673-687. 48. Dawe HR, Adams M, Wheway G, Szymanska K, Logan CV, Noegel AA, Gull K, Johnson CA: Nesprin-2 interacts with meckelin and mediates ciliogenesis via remodelling of the actin cytoskeleton. J Cell Sci 2009, 122:2716-2726. Provides further evidence of NE-independent nesprin function and implicates nesprins in plasma membrane coupling to the cytoskeleton. 49. Warren DT, Tajsic T, Mellad JA, Searles R, Zhang Q, Shanahan CM: Novel nuclear nesprin-2 variants tether active extracellular signal-regulated MAPKs 1 and 2 at promyelocytic leukaemia protein nuclear bodies and act to regulate smooth muscle cell proliferation. J Biol Chem 2009. 50. Seung Jin Han JHL, Ki Young Choi, Seung Hwan Hong: Novel p104 protein regulates cell proliferation through PI3K inhibition and p27Kip1 expression. BMB Rep 2009, 43:199-204.
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Nesprins LINC the nucleus and cytoskeleton Jason A Mellada, Derek T Warrena and Catherine M Shanahan Like other spectrin repeat proteins, nesprins co-ordinate and maintain cellular architecture by linking membranous organelles to the cytoskeleton. However nuclear envelope (NE) nesprins, uniquely hardwire the nuclear lamina to the cytoskeleton and molecular motors. Emerging evidence suggests that nesprins also form a continuous network linking the plasma membrane to the NE that potentially translates mechanical stimuli into nuclear reorganisation. Surprisingly, this network is also essential for cytoskeletal organisation and its disruption has dramatic effects on nuclear migration, centrosomal positioning, focal adhesion maturation and cell motility. Herein we review recent advances in our understanding of how nesprins couple to various filamentous systems within the cell and emphasise the importance of both KASH and KASH-less nesprin isoforms in these interactions. Address BHF Centre of Research Excellence, Division of Cardiovascular Medicine, King’s College London, SE5 9NU, UK a These authors contributed equally. Corresponding author: Shanahan, Catherine M ([email protected])
Current Opinion in Cell Biology 2011, 23:47–54 This review comes from a themed issue on Cell structure and dynamics Edited by Anne Ridley and Rebecca Heald Available online 20th December 2010 0955-0674/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2010.11.006
Introduction The nuclear envelope (NE) is a highly organised structure that partitions the nuclear and cytoplasmic environments of the cell. It is composed of two lipid bilayers, the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) and these are separated by a lumenal space [1,2]. Nuclear pore complexes span these membranes and regulate protein transport between the nucleoplasm and the cytosol [1,2]. NE structural and functional integrity is maintained via specific components of the INM and ONM that also physically link the cytoskeleton and nuclear lamina; a meshwork of intermediate filaments (IF) consisting of A-type and B-type lamins that underlie the INM (Figure 1) [3]. These essential connections enable the nucleus to maintain its position and tension within the cell during cell www.sciencedirect.com
polarisation, migration and differentiation and also relay mechanical signals to the nucleus [4]. The evolutionarily conserved, multi-isomeric Nuclear Envelope SPectRIN repeat proteins (nesprins) are an emerging family of nuclear-cytoplasmic couplers. Four genes (SYNE 1–4) encoding nesprins 1–4 have been identified so far. Nesprins are characterised by a central rod region of variable length, comprised of multiple spectrin repeats, and a C-terminal Klarsicht/ANC-1/Syne homology (KASH) transmembrane domain that facilitates NE localisation [5]. The nesprins are also characterised by variable N-terminal motifs that enable interactions with different components of the cytoskeleton. Giant isoforms of nesprin1 (1 MDa in size) and nesprin-2 (800 kDa) have Nterminal calponin homology (CH) domains that link to filamentous actin (F-actin), nesprin-3 (110 kDa) has a plectin-binding motif that permits interactions with cytoplasmic IFs while nesprin-4 interacts indirectly with microtubules [5–7]. Importantly, nesprins localise via the KASH domain to both the INM and ONM and are partitioned to these domains depending on their size and binding partners (Figure 1) [8]. INM nesprins-1/2 possess C-terminal spectrin repeats that can interact with both the INM protein emerin and A-type lamins [9,10]. Thus, nesprins can form structural connections on either face of the NE. Nesprins and other KASH-domain proteins are recruited to the NE by binding to the Sad1p-UNC84 (SUN) domain proteins Sun1 and Sun2 within the perinuclear space, forming the linker of the nucleoskeleton and cytoskeleton (LINC) complex (Figure 1) [8]. The LINC anchors the nuclear lamina, which binds INM Sun proteins, to the cytoskeleton via ONM nesprins. Overexpression of dominant-negative KASH domain constructs and knockdown of LINC components nesprins-1/2, Sun1/2 or lamin A uncouples the INM from the ONM, detaches the nucleus from the cytoskeleton and decreases mechanical stiffness [8]. The importance of the LINC in cytoskeletal organisation and biomechanical signalling is further reflected in the wide range of disease phenotypes resulting from mutations in nesprins-1/2, including cardiac and skeletal muscle defects in Emery Dreifuss muscular dystrophy (EDMD), cerebellar defects in autosomal recessive spinocerebellar ataxia 1 (ARCA1) and tendon contractures in autosomal recessive arthrogryposis multiplex congenita (AMC) [11–13].
Nesprins associate with multiple cytoskeletal systems The LINC has primarily been characterised as serving three functions: to tether the nucleus to the cytoskeleton, Current Opinion in Cell Biology 2011, 23:47–54
48 Cell structure and dynamics
Figure 1
Actin cytoskeleton Intermediate filaments
Microtubules Nesprin-4
Nesprin-3
Cytoplasm
Nesprins-1/2
Plectin
Kif5B
ONM Nuclear pore complex
SUN domain
SUN domain
SUN domain
SUN domain INM
Emerin Nucleoplasm
CH domain
Nuclear lamina
Spectrin repeat
KASH domain
SUN domain containing protein Current Opinion in Cell Biology
The LINC complex facilitates the coupling of the nuclear lamina to cytoplasmic cytoskeletal systems via the nesprins. The LINC complex is comprised of SUN domain containing proteins (SUN1 and 2, UNC84 and SPAG4) on the INM that bind to the nuclear lamina via interactions with lamin A, and potentially smaller nesprin-1/2 isoforms and emerin. The KASH domain of the larger isoforms of nesprins-1/2, nesprin-3 and nesprin-4 at the ONM associate with the SUN domain within the luminal space to tether the NE to either cytoplasmic actin, IFs or microtubule networks.
facilitate the mechanotransduction of peripheral signals to the nucleus and also to regulate cytoplasmic filament organisation during migration, adhesion and polarity establishment. It is clear that ONM nesprins are integral to all of these roles owing to their ability to indirectly or directly interact with all three major cytoplasmic filaments (Table 1). Connections with the actin cytoskeleton
The most characterised nesprin LINCs anchor the nucleus to the actin cytoskeleton. Surprisingly, the direct linkage of the NE to F-actin defines the mechanical properties of the entire cell via mechanotransduction between the cytoplasm and the nucleus [4]. Uncoupling of this mechanical link, in either nesprin-1 KASH domain or C-terminal spectrin repeat knockout mice, results in a loss of cellular tension that induces abnormal localisation of skeletal muscle nuclei and defects in strain transCurrent Opinion in Cell Biology 2011, 23:47–54
mission between the cell and nucleus [14,15]. Moreover, nuclear morphology is defined by thick actin bundles that are organised into a curved shell overlaying the nucleus, moulding it into a shape resembling overall cell morphology. Chemicals that depolymerise F-actin or inhibit Rho kinase, a key regulator of F-actin organisation and actomyosin contractility, release the nucleus from these physical constraints, resulting in nuclear morphology defects [16]. Similarly, disruption of the LINC complex, by either the expression of dominantnegative nesprin KASH constructs or mutation of nuclear lamina proteins, eliminates this actin shell and induces nuclear morphology defects [16]. Furthermore, uncoupling of the nucleus from the actin cytoskeleton triggers a loss of cellular tension. This loss of tension impacts on maturation of focal adhesions, important macromolecular cell–matrix adhesions that transmit mechanical force and other signals, as well as on cell motility. This suggests that www.sciencedirect.com
Nesprins LINC the nucleus and cytoskeleton Mellad, Warren and Shanahan 49
Table 1 Summary of demonstrated nesprin LINC complexes. Nesprins connect the NE to the cytoskeleton via direct interactions with F-actin or indirectly to microtubule and IF binding proteins. Connection Actin
F-actin
Nesprin
Function
Meckelin
Nesprin-1 Nesprin-2 Nesprin-2
IF
Lamin A Plectin
Nesprin-1/2 Nesprin-3
Chromatin organisation, NE architecture. NE-IF coupling
Microtubules
Kinesin-1 Kinesin-2 Dynein Dynactin
Nesprin-2/4 Nesprin-1 Nesprin-1/2 Nesprin-1/2
Nuclear migration, polarity. Vesicular transport. Nuclear migration, polarity. Nuclear migration, polarity
nuclear tethering to the actin cytoskeleton may be crucial for F-actin organisation throughout the cell [17,18,19]. Although the N-terminal paired CH domains remain the only characterised F-actin-binding module within nesprins-1/2 [20,21], evidence now exists for alternative connections to the actin cytoskeleton. For instance, nesprin-2 CH domain knockout dermal fibroblasts display nuclear blebbing and lobulations in vitro characteristic of LINC disruption. Interestingly, after several passages, these fibroblasts gradually express a novel, N-terminally truncated giant nesprin-2 isoform that alleviates this phenotype [22]. This suggests that similar to their homologues dystrophin and utrophin, nesprin spectrin repeats may also bind filamentous actin either directly or indirectly via as yet unknown binding partners [23–25]. Intriguingly, emerging data support the existence of Factin at the INM. Recent studies have shown that the nesprin binding partners emerin and lamin-A serve to stabilise and bundle F-actin [26,27]. Importantly, Factin may potentially contribute to LINC stability as emerin, lamin A and nesprin-1/2 spectrin repeats can all interact with the nucleoplasmic N-termini of Sun1/2 [28,29,30,31]. However, the nature and abundance of actin filaments at the INM remains controversial and further work is required to determine if INM nesprins and nuclear actin are also components of the LINC complex.
Connections with the centrosome/ microtubule network Nesprins also make multiple indirect connections with the microtubule network by forming microtubule motor LINCs. Analogous to the roles of UNC-83 and ZYG-12 in C. elegans and Klarsicht in Drosophila, these LINCs are essential for both nuclear and centrosomal positioning and therefore cell polarity [8]. Recent studies have demonstrated that nesprins-1/2 interact with dynein/ dynactin and kinesin-1 in the developing mouse brain and loss of these interactions in double nesprin-1/2KASH knockout mice uncouples the centrosome from www.sciencedirect.com
Nuclear positioning, Mechanotransduction. Ciliogenesis.
the neuronal nucleus resulting in severe nucleokinesis and interkinetic nuclear migration defects [30]. Similarly, zebrafish nesprin-2 also binds to dynactin and is necessary for photoreceptor interkinetic nuclear migration as demonstrated by knockdown and dominant-negative KASH overexpression studies [32]. Moreover, interactions between nesprin-4 and kinesin-1 regulate epithelial cell polarity and nuclear positioning with nesprin-4 overexpression dramatically displacing the centrosome and Golgi away from the NE [6]. Interestingly, a central spectrin repeat region of nesprin-1 also binds to the kinesin-2 subunit Kif3b and appears to play a role in cytokinesis and membrane transport. However whether these functions are mediated by a nesprin NE LINC or a KASH-less isoform (see below) must be explored further [33].
Nesprins associate with intermediate filament systems in the nucleus and cytoplasm Nesprins tether to IF systems in both the nucleus and cytoplasm and these connections potentially serve important organisational roles within the cell. The nuclear lamina is an IF meshwork and lamins-A/C were the first binding partners identified for nesprin-1 and nesprin-2. Our understanding of these complexes remains poor and it is not yet clear if these interactions are all LINC-dependent [5]. However both nesprins and lamins bind to multiple integral membrane proteins of the INM and one important function for these nuclear lamina complexes is the organisation of heterochromatin, which is facilitated by auxiliary proteins such as BAF, LAP2a and possibly smaller nesprin-1/2 isoforms [34,35]. Indeed, the dramatic heterochromatin reorganisation induced by knockdown of nesprins-1/2 in U2OS cells and fibroblasts may be partially attributed to the loss of INM isoforms and consequently disruption of a direct link between mechanical stimuli and chromatin organisation, although this possibility requires further investigation [12]. An emerging area for investigation is the interaction between nesprins and cytoplasmic IF networks. These LINCs are proposed to connect the NE to another form of Current Opinion in Cell Biology 2011, 23:47–54
50 Cell structure and dynamics
Figure 2
SR1
SR45
Nesprin-1 Giant 1β 1 1β 2 1α1 1α2
Drop1 CPG2
Nesprin-2 SR1
SR18
Giant 2γ 2β 1 2β 2 2α1 2α2
Nesprin-3
3α CH domain 3β
Spectrin repeat Nesprin-4 KASH domain
4 Current Opinion in Cell Biology
Previously published nesprin isoforms. LINC adaptability is increased by the generation of multiple nesprin isoforms of variable length and with different SR domains. The majority of isoforms characterised so far are N-terminal truncations that retain the KASH domain, suggesting that the proximity of cytoskeletal filaments and other nesprin-bound complexes to the NE may have functional significance. In addition, the number of characterised KASH-less isoforms is steadily increasing.
cell–matrix adhesion known as hemidesmosomes, providing further mechanical support for cells. For example, nesprin-3 binds to the CH domain of the IF-crosslinker plectin and thus forms a continuous IF network between the NE and hemidesmosomes [7,36]. Nesprin-3 appears to be central to IF LINC complex function as both plectin and keratins are drawn to the nuclear periphery of keratinocytes overexpressing nesprin3a [7]. It has been proposed that when actin filaments are naturally or artificially depolymerised the nesprin-3-dependent relocalisation of endogenous plectin to the nuclear periphery may facilitate rapid reorganisation of the cytoskeleton [7]. Current Opinion in Cell Biology 2011, 23:47–54
Different LINCs for different tissues The properties of a mechanically coupled, differentiated muscle cell are vastly different to a fibroblast or a polarised cell such as an endothelial cell. Thus, different cell types have unique biomechanical properties that are likely to require customised connections between the nucleus and cytoskeleton. It is likely that this specialisation is accomplished through the tissue-specific expression of nesprin genes and isoforms allowing cells to fine-tune these connections (Figure 2). For example, nesprin-4 is highly expressed only in specialised secretory epithelium where it is thought to be essential for www.sciencedirect.com
Nesprins LINC the nucleus and cytoskeleton Mellad, Warren and Shanahan 51
establishing centrosome positioning and cell polarity. Similarly, nesprin-1 is essential for neuromuscular junction (NMJ) formation and nesprin-2 is highly enriched in photoreceptor nuclei however the functional reasons for this specificity are not yet clear [6,15,30,32,37]. Nesprin isoforms also vary greatly between different tissues [38]. Nesprins 1/2 are especially abundant in muscle and express muscle specific isoforms. Moreover, mutations
in nesprin-1 and nesprin-2 have been reported in the muscle specific disease EDMD but these mutations appear to have little impact on other tissues. There is also a switch from larger to smaller NE nesprin isoforms during human myotube differentiation. This may serve to adapt LINC function enabling cells to change from a dynamic motile phenotype to a rigid, stable contractile phenotype [39]. It will be interesting to determine
Figure 3
Plasma membrane Binding partners? KASH domain containing isoforms?
KASH-less isoforms?
Actin IFs Microtubules
Cytoplasm
ONM Nuclear pore complex
SUN domain
SUN domain
SUN domain INM
Emerin
Nucleoplasm
Nuclear lamina
Actin filaments
CH domain
Spectrin repeat
KASH domain
SUN domain containing protein Current Opinion in Cell Biology
Nesprins form a mechanotransduction network between the plasma membrane and NE via connections with cytoplasmic cytoskeletal systems. Nesprins localise to the plasma membrane; whether or not these isoforms retain the KASH domain and how they are targeted remain unknown. The nesprin bridging network via the cytoskeleton potentially allows cells to maintain tension between the plasma membrane and nucleus that is essential for both organelle and cytoskeletal organisation within a cell [4]. www.sciencedirect.com
Current Opinion in Cell Biology 2011, 23:47–54
52 Cell structure and dynamics
whether LINCs are also able to dynamically respond to mechanical stimuli thus enabling the cell to rapidly adapt to different environmental conditions. The method of nesprin tethering to the NE may also contribute to LINC functional specialisation. Emerging evidence exists for non-canonical LINCs that do not solely rely on Sun1/2 or lamin A. For instance, Sun1/2 redundantly tether nesprin in some cell types but not in others [36,40,41,42] while torsin-1A, an ATPase localised to the perinuclear space, is also a KASH binding protein that regulates nesprin NE localisation [43,44]. LINCs have recently been implicated in highly specialised roles and have been shown to mediate chromosome pairing during meiosis in C. elegans. It will be important to determine if analogous specialised LINCs also operate in vertebrates [45].
Concluding remarks In conclusion, our knowledge of nesprin LINCs and their role in attaching the NE to the cytoskeleton has increased dramatically over the past few years. However, we still lack a complete understanding of the complexity of connections nesprins make with the filamentous cytoskeletal systems and identification of novel binding partners will help to address this deficit. Additionally, increasing evidence implicates the KASH containing nesprins as signalling scaffolds at the NE and potentially the KASH-less isoforms in other subcellular compartments. However, these KASH-less isoforms remain to be fully characterised. Indeed, a comprehensive read out of nesprin isoform profiles, localisations and interactions in different cell types will enable us to better understand both nesprin and LINC complex variability and function.
Acknowledgements Finally, in addition to their mechanical roles in LINC complex function, recent evidence suggests that nesprins may also serve as signalling scaffold proteins at the NE. Nesprin-2 has recently been shown to regulate WNT signalling and binds alpha and beta catenin at the NE, suggesting that in addition to their structural/mechanical roles LINC complexes may also function as signalling platforms [46].
This work was supported by British Heart Foundation (BHF) grants to CMS.
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27. Simon DN, Zastrow MS, Wilson KL: Direct actin binding to A and B-type lamin tails and actin filament bundling by the lamin A tail. Nucleus 2010, 1:1-9. Similar to the presence of intermediate filament networks both inside and outside the nucleus, this paper now reveals that F-actin exists on both faces of the NE. Surprisingly, lamins can directly bind and bundle F-actin at the INM. 28. Haque F, Lloyd DJ, Smallwood DT, Dent CL, Shanahan CM, Fry AM, Trembath RC, Shackleton S: SUN1 interacts with nuclear lamin A and cytoplasmic nesprins to provide a physical connection between the nuclear lamina and the cytoskeleton. Mol Cell Biol 2006, 26:3738-3751. 29. Haque F, Mazzeo D, Patel JT, Smallwood DT, Ellis JA, Shanahan CM, Shackleton S: Mammalian SUN protein interaction networks at the inner nuclear membrane and their role in laminopathy disease processes. J Biol Chem 2010, 285:3487-3498. Highlights the complexity of the LINC complex and describes novel INM interactions involving nesprin spectrin repeats, emerin and Sun1/2. 30. Zhang X, Lei K, Yuan X, Wu X, Zhuang Y, Xu T, Xu R, Han M: SUN1/ 2 and Syne/Nesprin-1/2 complexes connect centrosome to the nucleus during neurogenesis and neuronal migration in mice. Neuron 2009, 64:173-187. 31. Padmakumar VC, Libotte T, Lu W, Zaim H, Abraham S, Noegel AA, Gotzmann J, Foisner R, Karakesisoglou I: The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope. J Cell Sci 2005, 118:3419-3430. 32. Del Bene F, Wehman AM, Link BA, Baier H: Regulation of neurogenesis by interkinetic nuclear migration through an apical-basal notch gradient. Cell 2008, 134:1055-1065. 33. Fan J, Beck KA: A role for the spectrin superfamily member Syne-1 and kinesin II in cytokinesis. J Cell Sci 2004, 117:619-629. 34. Worman HJ, Courvalin JC: Nuclear envelope, nuclear lamina, and inherited disease. Int Rev Cytol 2005, 246:231-279. 35. Malik P, Zuleger N, Schirmer EC: Nuclear envelope influences on genome organization. Biochem Soc Trans 2010, 38:268-272. 36. Ketema M, Wilhelmsen K, Kuikman I, Janssen H, Hodzic D, Sonnenberg A: Requirements for the localization of nesprin-3 at the nuclear envelope and its interaction with plectin. J Cell Sci 2007, 120:3384-3394. 37. Puckelwartz MJ, Kessler E, Zhang Y, Hodzic D, Randles KN, Morris G, Earley JU, Hadhazy M, Holaska JM, Mewborn SK et al.: Disruption of nesprin-1 produces an Emery Dreifuss muscular dystrophy-like phenotype in mice. Hum Mol Genet 2009, 18:607-620. 38. Zhang Q, Skepper JN, Yang F, Davies JD, Hegyi L, Roberts RG, Weissberg PL, Ellis JA, Shanahan CM: Nesprins: a novel family of spectrin-repeat-containing proteins that localize to the nuclear membrane in multiple tissues. J Cell Sci 2001, 114:4485-4498. 39. Randles KN, Lam le T, Sewry CA, Puckelwartz M, Furling D, Wehnert M, McNally EM, Morris GE: Nesprins, but not sun proteins, switch isoforms at the nuclear envelope during muscle development. Dev Dyn 2010, 239:998-1009. The first in-depth study of changes in nesprin isoform expression during muscle development. 40. Ostlund C, Folker ES, Choi JC, Gomes ER, Gundersen GG, Worman HJ: Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins. J Cell Sci 2009. An elegant description of LINC complex stability at the NE using FRAP, showing that these complexes are fluid. 41. Crisp M, Liu Q, Roux K, Rattner JB, Shanahan C, Burke B, Stahl PD, Hodzic D: Coupling of the nucleus and cytoplasm: role of the LINC complex. J Cell Biol 2006, 172:41-53. 42. Lei K, Zhang X, Ding X, Guo X, Chen M, Zhu B, Xu T, Zhuang Y, Xu R, Han M: SUN1 and SUN2 play critical but partially redundant roles in anchoring nuclei in skeletal muscle cells in mice. Proc Natl Acad Sci USA 2009, 106:10207-10212. Current Opinion in Cell Biology 2011, 23:47–54
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43. Vander Heyden AB, Naismith TV, Snapp EL, Hodzic D, Hanson PI: LULL1 retargets TorsinA to the nuclear envelope revealing an activity that is impaired by the DYT1 dystonia mutation. Mol Biol Cell 2009, 20:2661-2672. 44. Nery FC, Zeng J, Niland BP, Hewett J, Farley J, Irimia D, Li Y, Wiche G, Sonnenberg A, Breakefield XO: TorsinA binds the KASH domain of nesprins and participates in linkage between nuclear envelope and cytoskeleton. J Cell Sci 2008, 121:3476-3486. 45. Penkner A, Tang L, Novatchkova M, Ladurner M, Fridkin A, Gruenbaum Y, Schweizer D, Loidl J, Jantsch V: The nuclear envelope protein Matefin/SUN-1 is required for homologous pairing in C. elegans meiosis. Dev Cell 2007, 12:873-885. 46. Neumann S, Schneider M, Daugherty RL, Gottardi CJ, Eming SA, Beijer A, Noegel AA, Karakesisoglou I: Nesprin-2 interacts with {alpha}-catenin and regulates Wnt signaling at the nuclear envelope. J Biol Chem 2010, 285:34932-34938. 47. Zhang Q, Ragnauth CD, Skepper JN, Worth NF, Warren DT, Roberts RG, Weissberg PL, Ellis JA, Shanahan CM: Nesprin-2 is a
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multi-isomeric protein that binds lamin and emerin at the nuclear envelope and forms a subcellular network in skeletal muscle. J Cell Sci 2005, 118:673-687. 48. Dawe HR, Adams M, Wheway G, Szymanska K, Logan CV, Noegel AA, Gull K, Johnson CA: Nesprin-2 interacts with meckelin and mediates ciliogenesis via remodelling of the actin cytoskeleton. J Cell Sci 2009, 122:2716-2726. Provides further evidence of NE-independent nesprin function and implicates nesprins in plasma membrane coupling to the cytoskeleton. 49. Warren DT, Tajsic T, Mellad JA, Searles R, Zhang Q, Shanahan CM: Novel nuclear nesprin-2 variants tether active extracellular signal-regulated MAPKs 1 and 2 at promyelocytic leukaemia protein nuclear bodies and act to regulate smooth muscle cell proliferation. J Biol Chem 2009. 50. Seung Jin Han JHL, Ki Young Choi, Seung Hwan Hong: Novel p104 protein regulates cell proliferation through PI3K inhibition and p27Kip1 expression. BMB Rep 2009, 43:199-204.
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