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Catherine M Shanahan - One of the best experts on this subject based on the ideXlab platform.
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nesprin 1 2 roles in nuclear envelope organisation myogenesis and muscle disease
Biochemical Society Transactions, 2018Co-Authors: Li Rao, Catherine M Shanahan, Can Zhou, Qiuping ZhangAbstract:Nesprins (nuclear envelope Spectrin Repeat proteins) are multi-isomeric scaffolding proteins. Nesprin-1 and -2 are highly expressed in skeletal and cardiac muscles and together with SUN (Sad1p/UNC84) domain-containing proteins form the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex at the nuclear envelope in association with lamin A/C and emerin. Mutations in nesprin-1/2 have been found in patients with autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). Several lines of evidence indicate that compromised LINC complex function is the critical step leading to muscle disease. Here, we review recent advances in our understanding of the functions of nesprin-1/2 in the LINC complex and mechanistic insights into how mutations in nesprin-1/2 lead to nesprin-related muscle diseases, in particular DCM and EDMD.
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identification of novel nesprin 1 binding partners and cytoplasmic matrin 3 in processing bodies
Molecular Biology of the Cell, 2016Co-Authors: Dipen Rajgor, Jonathan G Hanley, Catherine M ShanahanAbstract:Nesprins are highly conserved Spectrin Repeat-containing scaffold proteins predominantly known to function at the nuclear envelope (NE). However, nesprin isoforms are emerging with localizations and scaffolding functions at sites away from the NE, suggesting their functions are more diverse than originally thought. In this study, we combined nesprin-1 coimmunoprecipitations with mass spectrometry to identify novel nesprin-1 binding partners for isoforms that localize to subcellular compartments beyond the NE. We show that one of these interactors, matrin-3 (matr3), localizes to mRNA processing bodies (PBs), where we have previously shown a nesprin-1 isoform to localize. Furthermore, we show that Matr3 is part of PB mRNP complexes, is a regulator of miRNA-mediated gene silencing, and possibly shuttles to stress granules in stressed cells. More importantly, we identify a new C-terminally truncated Matr3 isoform that is likely to be involved in these functions and PB localization. This study highlights several novel nesprin-1 binding partners and a new function and localization for Matr3 in cytoplasmic RNA granules.
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Multiple Novel Nesprin-1 and Nesprin-2 Variants Act as Versatile Tissue-Specific Intracellular Scaffolds
2016Co-Authors: Dipen Rajgor, Qiuping Zhang, Jason A Mellad, Flavia Autore, Catherine M ShanahanAbstract:Background: Nesprins (Nuclear envelope Spectrin-Repeat proteins) are a novel family of giant Spectrin-Repeat containing proteins. The nesprin-1 and nesprin-2 genes consist of 146 and 116 exons which encode proteins of,1mDa and,800 kDa is size respectively when all the exons are utilised in translation. However emerging data suggests that the nesprins have multiple alternative start and termination sites throughout their genes allowing the generation of smaller isoforms. Results: In this study we set out to identify novel alternatively transcribed nesprin variants by screening the EST database and by using RACE analysis to identify cDNA ends. These two methods provided potential hits for alternative start and termination sites that were validated by PCR and DNA sequencing. We show that these alternative sites are not only expressed in a tissue specific manner but by combining different sites together it is possible to create a wide array of nesprin variants. By cloning and expressing small novel nesprin variants into human fibroblasts and U2OS cells we show localization to actin stress-fibres, focal adhesions, microtubules, the nucleolus, nuclear matrix and the nuclear envelope (NE). Furthermore we show that the sub-cellular localization of individual nesprin variants can vary depending on the cell type, suggesting any single nesprin variant may have different functions in different cell types. Conclusions: These studies suggest nesprins act as highly versatile tissue specific intracellular protein scaffolds and identify potential novel functions for nesprins beyond cytoplasmic-nuclear coupling. These alternate functions may also account fo
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mammalian microtubule p body dynamics are mediated by nesprin 1
Journal of Cell Biology, 2014Co-Authors: Dipen Rajgor, Jason A Mellad, Daniel Soong, Jerome B Rattner, Marvin J Fritzler, Catherine M ShanahanAbstract:Nesprins are a multi-isomeric family of Spectrin-Repeat (SR) proteins, predominantly known as nuclear envelope scaffolds. However, isoforms that function beyond the nuclear envelope remain poorly examined. Here, we characterize p50Nesp1, a 50-kD isoform that localizes to processing bodies (PBs), where it acts as a microtubule-associated protein capable of linking mRNP complexes to microtubules. Overexpression of dominant-negative p50Nesp1 caused Rck/p54, but not GW182, displacement from microtubules, resulting in reduced PB movement and cross talk with stress granules (SGs). These cells disassembled canonical SGs induced by sodium arsenite, but not those induced by hydrogen peroxide, leading to cell death and revealing PB–microtubule attachment is required for hydrogen peroxide-induced SG anti-apoptotic functions. Furthermore, p50Nesp1 was required for miRNA-mediated silencing and interacted with core miRISC silencers Ago2 and Rck/p54 in an RNA-dependent manner and with GW182 in a microtubule-dependent manner. These data identify p50Nesp1 as a multi-functional PB component and microtubule scaffold necessary for RNA granule dynamics and provides evidence for PB and SG micro-heterogeneity.
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Large-Scale Modelling of the Divergent Spectrin Repeats in Nesprins: Giant Modular Proteins
2013Co-Authors: Flavia Autore, Catherine M Shanahan, Roland G Roberts, Mark Pfuhl, Xueping Quan, Aisling Williams, Franca FraternaliAbstract:Nesprin-1 and nesprin-2 are nuclear envelope (NE) proteins characterized by a common structure of an SR (Spectrin Repeat) rod domain and a C-terminal transmembrane KASH [Klarsicht–ANC–Syne-homology] domain and display N-terminal actin-binding CH (calponin homology) domains. Mutations in these proteins have been described in Emery-Dreifuss muscular dystrophy and attributed to disruptions of interactions at the NE with nesprins binding partners, lamin A/C and emerin. Evolutionary analysis of the rod domains of the nesprins has shown that they are almost entirely composed of unbroken SR-like structures. We present a bioinformatical approach to accurate definition of the boundaries of each SR by comparison with canonical SR structures, allowing for a large-scale homology modelling of the 74 nesprin-1 and 56 nesprin-2 SRs. The exposed and evolutionary conserved residues identify important pbs for protein-protein interactions that can guide tailored binding experiments. Most importantly, the bioinformatics analyses and the 3D models have been central to the design of selected constructs for protein expression. 1D NMR and CD spectra have been performed of the expressed SRs, showing a folded, stable, high content α-helical structure, typical of SRs. Molecular Dynamics simulations have been performed to study the structural and elastic properties of consecutive SRs, revealing insights in the mechanical properties adopted by these modules in the cell.
Qiuping Zhang - One of the best experts on this subject based on the ideXlab platform.
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nesprin 1 2 roles in nuclear envelope organisation myogenesis and muscle disease
Biochemical Society Transactions, 2018Co-Authors: Li Rao, Catherine M Shanahan, Can Zhou, Qiuping ZhangAbstract:Nesprins (nuclear envelope Spectrin Repeat proteins) are multi-isomeric scaffolding proteins. Nesprin-1 and -2 are highly expressed in skeletal and cardiac muscles and together with SUN (Sad1p/UNC84) domain-containing proteins form the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex at the nuclear envelope in association with lamin A/C and emerin. Mutations in nesprin-1/2 have been found in patients with autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). Several lines of evidence indicate that compromised LINC complex function is the critical step leading to muscle disease. Here, we review recent advances in our understanding of the functions of nesprin-1/2 in the LINC complex and mechanistic insights into how mutations in nesprin-1/2 lead to nesprin-related muscle diseases, in particular DCM and EDMD.
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Multiple Novel Nesprin-1 and Nesprin-2 Variants Act as Versatile Tissue-Specific Intracellular Scaffolds
2016Co-Authors: Dipen Rajgor, Qiuping Zhang, Jason A Mellad, Flavia Autore, Catherine M ShanahanAbstract:Background: Nesprins (Nuclear envelope Spectrin-Repeat proteins) are a novel family of giant Spectrin-Repeat containing proteins. The nesprin-1 and nesprin-2 genes consist of 146 and 116 exons which encode proteins of,1mDa and,800 kDa is size respectively when all the exons are utilised in translation. However emerging data suggests that the nesprins have multiple alternative start and termination sites throughout their genes allowing the generation of smaller isoforms. Results: In this study we set out to identify novel alternatively transcribed nesprin variants by screening the EST database and by using RACE analysis to identify cDNA ends. These two methods provided potential hits for alternative start and termination sites that were validated by PCR and DNA sequencing. We show that these alternative sites are not only expressed in a tissue specific manner but by combining different sites together it is possible to create a wide array of nesprin variants. By cloning and expressing small novel nesprin variants into human fibroblasts and U2OS cells we show localization to actin stress-fibres, focal adhesions, microtubules, the nucleolus, nuclear matrix and the nuclear envelope (NE). Furthermore we show that the sub-cellular localization of individual nesprin variants can vary depending on the cell type, suggesting any single nesprin variant may have different functions in different cell types. Conclusions: These studies suggest nesprins act as highly versatile tissue specific intracellular protein scaffolds and identify potential novel functions for nesprins beyond cytoplasmic-nuclear coupling. These alternate functions may also account fo
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multiple novel nesprin 1 and nesprin 2 variants act as versatile tissue specific intracellular scaffolds
PLOS ONE, 2012Co-Authors: Dipen Rajgor, Qiuping Zhang, Jason A Mellad, Flavia Autore, Catherine M ShanahanAbstract:Background Nesprins (Nuclear envelope Spectrin-Repeat proteins) are a novel family of giant Spectrin-Repeat containing proteins. The nesprin-1 and nesprin-2 genes consist of 146 and 116 exons which encode proteins of ∼1mDa and ∼800 kDa is size respectively when all the exons are utilised in translation. However emerging data suggests that the nesprins have multiple alternative start and termination sites throughout their genes allowing the generation of smaller isoforms.
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Multiple Novel Nesprin-1 and Nesprin-2 Variants Act as Versatile Tissue-Specific Intracellular Scaffolds
2012Co-Authors: Dipen Rajgor, Qiuping Zhang, Jason A Mellad, Flavia Autore, Catherine M ShanahanAbstract:BackgroundNesprins (Nuclear envelope Spectrin-Repeat proteins) are a novel family of giant Spectrin-Repeat containing proteins. The nesprin-1 and nesprin-2 genes consist of 146 and 116 exons which encode proteins of ∼1mDa and ∼800 kDa is size respectively when all the exons are utilised in translation. However emerging data suggests that the nesprins have multiple alternative start and termination sites throughout their genes allowing the generation of smaller isoforms. ResultsIn this study we set out to identify novel alternatively transcribed nesprin variants by screening the EST database and by using RACE analysis to identify cDNA ends. These two methods provided potential hits for alternative start and termination sites that were validated by PCR and DNA sequencing. We show that these alternative sites are not only expressed in a tissue specific manner but by combining different sites together it is possible to create a wide array of nesprin variants. By cloning and expressing small novel nesprin variants into human fibroblasts and U2OS cells we show localization to actin stress-fibres, focal adhesions, microtubules, the nucleolus, nuclear matrix and the nuclear envelope (NE). Furthermore we show that the sub-cellular localization of individual nesprin variants can vary depending on the cell type, suggesting any single nesprin variant may have different functions in different cell types. ConclusionsThese studies suggest nesprins act as highly versatile tissue specific intracellular protein scaffolds and identify potential novel functions for nesprins beyond cytoplasmic-nuclear coupling. These alternate functions may also account for the diverse range of disease phenotypes observed when these genes are mutated.
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nesprin 1 and actin contribute to nuclear and cytoskeletal defects in lamin a c deficient cardiomyopathy
Journal of Molecular and Cellular Cardiology, 2011Co-Authors: Vesna Nikolovakrstevski, Qiuping Zhang, Christiana Leimena, Xiaohui Xiao, Scott H Kesteven, Juchiat Tan, Li Sze Yeo, Arthur Carlton, Stewart I Head, Catherine M ShanahanAbstract:Lamin A/C mutations are the most common cause of familial dilated cardiomyopathy (DCM) but the pathogenetic mechanisms are incompletely understood. Nesprins are Spectrin Repeat-containing proteins that interact with lamin A/C and are components of the linker-of-nucleoskeleton-and-cytoskeleton (LINC) complex that connects the nuclear envelope to the actin cytoskeleton. Our aim was to determine whether changes in nesprin-1 and actin might contribute to DCM in homozygous Lmna knockout ( Lmna −/− ) mice. Here we find that Lmna −/ − cardiomyocytes have altered nuclear envelope morphology, disorganization of nesprin-1 and heterogeneity in the distribution of nuclear and cytoskeletal actin. Functional interactions of nesprin-1 with nuclear G-actin and with the cytoskeletal γ-actin, α-cardiac actin and α-smooth muscle actin (α-SMA) isoforms were shown by immunoprecipitation and Western blotting. At 4–6 weeks of age, Lmna −/ − mice had normal levels of γ-actin and α-cardiac actin, but α-SMA expression was increased by 50%. In contrast to the predominant vascular distribution of α-SMA in WT ventricular sections, α-SMA had a diffuse staining pattern in Lmna −/ − sections. Osmotic swelling studies showed enhanced radial swelling in Lmna −/ − cardiomyocytes indicative of cytoskeletal instability. The distensibility of Lmna −/ − cardiomyocytes with osmotic stress was reduced by addition of α-SMA-specific fusion peptide. Our findings support a model in which uncoupling of the nucleus and cytoskeleton associated with disruption of the LINC complex promotes mechanical instability and defective force transmission in cardiomyocytes. Changes in the distribution and expression patterns of nuclear and cytoskeletal actin suggest that diverse transcriptional and structural defects may also contribute to DCM in Lmna −/ − mice.
Arnoud Sonnenberg - One of the best experts on this subject based on the ideXlab platform.
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the structure of the plakin domain of plectin reveals a non canonical sh3 domain interacting with its fourth Spectrin Repeat
Journal of Biological Chemistry, 2011Co-Authors: Esther Ortega, Arnoud Sonnenberg, Ruben M Buey, Jose M De PeredaAbstract:Abstract Plectin belongs to the plakin family of cytoskeletal crosslinkers, which is part of the Spectrin superfamily. Plakins contain an N-terminal conserved region, the plakin domain, which is formed by an array of Spectrin Repeats (SR) and a Src-homology 3 (SH3), and harbors binding sites for junctional proteins. We have combined x-ray crystallography and small angle x-ray scattering (SAXS) to elucidate the structure of the central region of the plakin domain of plectin, which corresponds to the SR3, SR4, SR5, and SH3 domains. The crystal structures of the SR3-SR4 and SR4-SR5-SH3 fragments were determined to 2.2 and 2.95 A resolution, respectively. The SH3 of plectin presents major alterations as compared with canonical Pro-rich binding SH3 domains, suggesting that plectin does not recognize Pro-rich motifs. In addition, the SH3 binding site is partially occluded by an intramolecular contact with the SR4. Residues of this pseudo-binding site and the SR4/SH3 interface are conserved within the plakin family, suggesting that the structure of this part of the plectin molecule is similar to that of other plakins. We have created a model for the SR3-SR4-SR5-SH3 region, which agrees well with SAXS data in solution. The three SRs form a semi-flexible rod that is not altered by the presence of the SH3 domain, and it is similar to those found in Spectrins. The flexibility of the plakin domain, in analogy with Spectrins, might contribute to the role of plakins in maintaining the stability of tissues subject to mechanical stress.
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nesprin 3 augments peripheral nuclear localization of intermediate filaments in zebrafish
Journal of Cell Science, 2011Co-Authors: Ruben Postel, Mirjam Ketema, Ingrid Kuikman, Jose M De Pereda, Arnoud SonnenbergAbstract:The outer nuclear membrane protein nesprin-3 binds the cytoskeletal linker protein plectin, which are proposed to anchor the intermediate filaments to the nuclear envelope. To investigate the function of nesprin-3 in vivo, we used the zebrafish as a vertebrate model system. Zebrafish nesprin-3 is expressed at the nuclear envelope of epidermal and skeletal muscle cells during development. Unexpectedly, loss of nesprin-3 did not affect embryonic development, viability or fertility. However, nesprin-3-deficient zebrafish embryos showed a reduced concentration of intermediate filaments around the nucleus. Additional analysis revealed the presence of two nesprin-3 isoforms in zebrafish, nesprin-3α and nesprin-3β. Nesprin-3β is only expressed during early development and lacks seven amino acids in its first Spectrin Repeat that are crucial for plectin binding and recruitment to the nuclear envelope. These seven amino acids are highly conserved and we showed that residues R43 and L44 within this motif are required for plectin binding. Furthermore, several residues in the actin-binding domain of plectin that are crucial for binding to the integrin β4 subunit are also important for the binding to nesprin-3α, indicating partial overlapping binding sequences for nesprin-3α and integrin β4. All this shows that nesprin-3 is dispensable for normal development in zebrafish, but important for mediating the association of the intermediate filament system with the nucleus in vivo.
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the structure of a tandem pair of Spectrin Repeats of plectin reveals a modular organization of the plakin domain
Journal of Molecular Biology, 2007Co-Authors: Arnoud Sonnenberg, Ana M Rojas, Jose M De PeredaAbstract:Plectin is a large and versatile cytoskeletal linker and member of the plakin protein family. Plakins share a conserved region called the plakin domain located near their N terminus. We have determined the crystal structure of an N-terminal fragment of the plakin domain of plectin to 2.05 A resolution. This region is adjacent to the actin-binding domain and is required for efficient binding to the integrin α6β4 in hemidesmosomes. The structure is formed by two Spectrin Repeats connected by an α-helix that spans these two Repeats. While the first Repeat is very similar to other known structures, the second Repeat is structurally different with a hydrophobic core, narrower than that in canonical Spectrin Repeats. Sequence analysis of the plakin domain revealed the presence of up to nine consecutive Spectrin Repeats organized in an array of tandem modules, and a Src-homology 3 domain inserted in the central Spectrin Repeat. The structure of the plakin domain is reminiscent of the modular organization of members of the Spectrin family. The architecture of the plakin domain suggests that it forms an elongated and flexible structure, and provides a novel molecular explanation for the contribution of plectin and other plakins to the elasticity and stability of tissues subjected to mechanical stress, such as the skin and striated muscle.
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nesprin 3 a novel outer nuclear membrane protein associates with the cytoskeletal linker protein plectin
Journal of Cell Biology, 2005Co-Authors: Kevin Wilhelmsen, Ingrid Kuikman, Hans Janssen, Sandy H M Litjens, Ntambua Tshimbalanga, Iman Van Den Bout, Karine Raymond, Arnoud SonnenbergAbstract:Despite their importance in cell biology, the mechanisms that maintain the nucleus in its proper position in the cell are not well understood. This is primarily the result of an incomplete knowledge of the proteins in the outer nuclear membrane (ONM) that are able to associate with the different cytoskeletal systems. Two related ONM proteins, nuclear envelope Spectrin Repeat (nesprin)–1 and –2, are known to make direct connections with the actin cytoskeleton through their NH2-terminal actin-binding domain (ABD). We have now isolated a third member of the nesprin family that lacks an ABD and instead binds to the plakin family member plectin, which can associate with the intermediate filament (IF) system. Overexpression of nesprin-3 results in a dramatic recruitment of plectin to the nuclear perimeter, which is where these two molecules are colocalized with both keratin-6 and -14. Importantly, plectin binds to the integrin α6β4 at the cell surface and to nesprin-3 at the ONM in keratinocytes, suggesting that there is a continuous connection between the nucleus and the extracellular matrix through the IF cytoskeleton.
Hiroyuki Tomimitsu - One of the best experts on this subject based on the ideXlab platform.
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an autosomal dominant cerebellar ataxia linked to chromosome 16q22 1 is associated with a single nucleotide substitution in the 5 untranslated region of the gene encoding a protein with Spectrin Repeat and rho guanine nucleotide exchange factor domains
American Journal of Human Genetics, 2005Co-Authors: Kinya Ishikawa, Shuta Toru, Taiji Tsunemi, Kazuhiro Kobayashi, Takanori Yokota, Takeshi Amino, Kiyoshi Owada, Hiroto Fujigasaki, Masaki Sakamoto, Hiroyuki TomimitsuAbstract:Autosomal dominant cerebellar ataxia (ADCA) is a group of heterogeneous neurodegenerative disorders. By positional cloning, we have identified the gene strongly associated with a form of degenerative ataxia (chromosome 16q22.1–linked ADCA) that clinically shows progressive pure cerebellar ataxia. Detailed examination by use of audiogram suggested that sensorineural hearing impairment may be associated with ataxia in our families. After restricting the candidate region in chromosome 16q22.1 by haplotype analysis, we found that all patients from 52 unrelated Japanese families harbor a heterozygous C→T single-nucleotide substitution, 16 nt upstream of the putative translation initiation site of the gene for a hypothetical protein DKFZP434I216, which we have called “puratrophin-1” (Purkinje cell atrophy associated protein-1). The full-length puratrophin-1 mRNA had an open reading frame of 3,576 nt, predicted to contain important domains, including the Spectrin Repeat and the guanine-nucleotide exchange factor (GEF) for Rho GTPases, followed by the Dbl-homologous domain, which indicates the role of puratrophin-1 in intracellular signaling and actin dynamics at the Golgi apparatus. Puratrophin-1—normally expressed in a wide range of cells, including epithelial hair cells in the cochlea—was aggregated in Purkinje cells of the chromosome 16q22.1–linked ADCA brains. Consistent with the protein prediction data of puratrophin-1, the Golgi-apparatus membrane protein and Spectrin also formed aggregates in Purkinje cells. The present study highlights the importance of the 5′ untranslated region (UTR) in identification of genes of human disease, suggests that a single-nucleotide substitution in the 5′ UTR could be associated with protein aggregation, and indicates that the GEF protein is associated with cerebellar degeneration in humans.
Jose M De Pereda - One of the best experts on this subject based on the ideXlab platform.
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the structure of the plakin domain of plectin reveals a non canonical sh3 domain interacting with its fourth Spectrin Repeat
Journal of Biological Chemistry, 2011Co-Authors: Esther Ortega, Arnoud Sonnenberg, Ruben M Buey, Jose M De PeredaAbstract:Abstract Plectin belongs to the plakin family of cytoskeletal crosslinkers, which is part of the Spectrin superfamily. Plakins contain an N-terminal conserved region, the plakin domain, which is formed by an array of Spectrin Repeats (SR) and a Src-homology 3 (SH3), and harbors binding sites for junctional proteins. We have combined x-ray crystallography and small angle x-ray scattering (SAXS) to elucidate the structure of the central region of the plakin domain of plectin, which corresponds to the SR3, SR4, SR5, and SH3 domains. The crystal structures of the SR3-SR4 and SR4-SR5-SH3 fragments were determined to 2.2 and 2.95 A resolution, respectively. The SH3 of plectin presents major alterations as compared with canonical Pro-rich binding SH3 domains, suggesting that plectin does not recognize Pro-rich motifs. In addition, the SH3 binding site is partially occluded by an intramolecular contact with the SR4. Residues of this pseudo-binding site and the SR4/SH3 interface are conserved within the plakin family, suggesting that the structure of this part of the plectin molecule is similar to that of other plakins. We have created a model for the SR3-SR4-SR5-SH3 region, which agrees well with SAXS data in solution. The three SRs form a semi-flexible rod that is not altered by the presence of the SH3 domain, and it is similar to those found in Spectrins. The flexibility of the plakin domain, in analogy with Spectrins, might contribute to the role of plakins in maintaining the stability of tissues subject to mechanical stress.
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nesprin 3 augments peripheral nuclear localization of intermediate filaments in zebrafish
Journal of Cell Science, 2011Co-Authors: Ruben Postel, Mirjam Ketema, Ingrid Kuikman, Jose M De Pereda, Arnoud SonnenbergAbstract:The outer nuclear membrane protein nesprin-3 binds the cytoskeletal linker protein plectin, which are proposed to anchor the intermediate filaments to the nuclear envelope. To investigate the function of nesprin-3 in vivo, we used the zebrafish as a vertebrate model system. Zebrafish nesprin-3 is expressed at the nuclear envelope of epidermal and skeletal muscle cells during development. Unexpectedly, loss of nesprin-3 did not affect embryonic development, viability or fertility. However, nesprin-3-deficient zebrafish embryos showed a reduced concentration of intermediate filaments around the nucleus. Additional analysis revealed the presence of two nesprin-3 isoforms in zebrafish, nesprin-3α and nesprin-3β. Nesprin-3β is only expressed during early development and lacks seven amino acids in its first Spectrin Repeat that are crucial for plectin binding and recruitment to the nuclear envelope. These seven amino acids are highly conserved and we showed that residues R43 and L44 within this motif are required for plectin binding. Furthermore, several residues in the actin-binding domain of plectin that are crucial for binding to the integrin β4 subunit are also important for the binding to nesprin-3α, indicating partial overlapping binding sequences for nesprin-3α and integrin β4. All this shows that nesprin-3 is dispensable for normal development in zebrafish, but important for mediating the association of the intermediate filament system with the nucleus in vivo.
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Nesprin-3 augments peripheral nuclear localization of intermediate filaments in zebrafish
'The Company of Biologists', 2011Co-Authors: Jose M De PeredaAbstract:10 páginas, 5 figuras.-- et al.The outer nuclear membrane protein nesprin-3 binds the cytoskeletal linker protein plectin, which are proposed to anchor the intermediate filaments to the nuclear envelope. To investigate the function of nesprin-3 in vivo, we used the zebrafish as a vertebrate model system. Zebrafish nesprin-3 is expressed at the nuclear envelope of epidermal and skeletal muscle cells during development. Unexpectedly, loss of nesprin-3 did not affect embryonic development, viability or fertility. However, nesprin-3-deficient zebrafish embryos showed a reduced concentration of intermediate filaments around the nucleus. Additional analysis revealed the presence of two nesprin-3 isoforms in zebrafish, nesprin-3α and nesprin-3β. Nesprin-3β is only expressed during early development and lacks seven amino acids in its first Spectrin Repeat that are crucial for plectin binding and recruitment to the nuclear envelope. These seven amino acids are highly conserved and we showed that residues R43 and L44 within this motif are required for plectin binding. Furthermore, several residues in the actin-binding domain of plectin that are crucial for binding to the integrin β4 subunit are also important for the binding to nesprin-3α, indicating partial overlapping binding sequences for nesprin-3α and integrin β4. All this shows that nesprin-3 is dispensable for normal development in zebrafish, but important for mediating the association of the intermediate filament system with the nucleus in vivo.Peer reviewe
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the structure of a tandem pair of Spectrin Repeats of plectin reveals a modular organization of the plakin domain
Journal of Molecular Biology, 2007Co-Authors: Arnoud Sonnenberg, Ana M Rojas, Jose M De PeredaAbstract:Plectin is a large and versatile cytoskeletal linker and member of the plakin protein family. Plakins share a conserved region called the plakin domain located near their N terminus. We have determined the crystal structure of an N-terminal fragment of the plakin domain of plectin to 2.05 A resolution. This region is adjacent to the actin-binding domain and is required for efficient binding to the integrin α6β4 in hemidesmosomes. The structure is formed by two Spectrin Repeats connected by an α-helix that spans these two Repeats. While the first Repeat is very similar to other known structures, the second Repeat is structurally different with a hydrophobic core, narrower than that in canonical Spectrin Repeats. Sequence analysis of the plakin domain revealed the presence of up to nine consecutive Spectrin Repeats organized in an array of tandem modules, and a Src-homology 3 domain inserted in the central Spectrin Repeat. The structure of the plakin domain is reminiscent of the modular organization of members of the Spectrin family. The architecture of the plakin domain suggests that it forms an elongated and flexible structure, and provides a novel molecular explanation for the contribution of plectin and other plakins to the elasticity and stability of tissues subjected to mechanical stress, such as the skin and striated muscle.