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Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin.

Wilhelmsen K, Litjens SH, Kuikman I, Tshimbalanga N, Janssen H, van den Bout I, Raymond K, Sonnenberg A - J. Cell Biol. (2005)

Bottom Line: 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.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 alpha6beta4 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
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 alpha6beta4 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.

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Sequence analysis of nesprin-3. (A) Scaled diagrams of the nesprin-1, -2, -3α, and -3β, ANC-1, UNC-83, and D. melanogaster Klarsicht protein are presented. The sequence alignment of the KASH domain of the different murine nesprins, ANC-1 and UNC-83, with the conserved domain in the D. melanogaster Klarsicht protein is shown with similar amino acids shaded gray. (B) Organization of the nesprin-3 genome. The 18 exons are labeled as E1–E18 and are depicted as closed bars, except the exon unique to nesprin-3α (E3), which is depicted as an open box. The translation start sites for nesprin-3α and -3β are shown in E2. The distances are drawn to scale with the measurement bar. (C) SRs 1, 3, 5, and 7, and 2, 4, 6, and 8 of nesprin-3α are shown aligned with the 13th and 14th SRs of spectrin-β2, respectively. Dark gray boxes depict alignment of more than three similar amino acids in an alignment set. Light gray boxes depict similar amino acids to a majority of those in the opposing alignment set. A diamond indicates that residues are conserved in only one alignment set, and an asterisk indicates residues that are conserved in both sets. Below the SR alignment the structures of nesprin-3α and -3β are shown. The location of the exons are denoted by bars and labeled in between each accordingly as E1–E18. The region of nesprin-3α encoded by the cDNA found in the Y2H screen is underlined, as is the sequence for nesprin-3β (RIKEN clone 4831426I19) found in the database. The SRs are depicted as open boxes. In exon 18, the RIKEN sequence continues for another 2,198 nucleotides after the stop codon. The NH2 terminus is to the left and the COOH terminus is to the right. The distances are drawn to scale.
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fig2: Sequence analysis of nesprin-3. (A) Scaled diagrams of the nesprin-1, -2, -3α, and -3β, ANC-1, UNC-83, and D. melanogaster Klarsicht protein are presented. The sequence alignment of the KASH domain of the different murine nesprins, ANC-1 and UNC-83, with the conserved domain in the D. melanogaster Klarsicht protein is shown with similar amino acids shaded gray. (B) Organization of the nesprin-3 genome. The 18 exons are labeled as E1–E18 and are depicted as closed bars, except the exon unique to nesprin-3α (E3), which is depicted as an open box. The translation start sites for nesprin-3α and -3β are shown in E2. The distances are drawn to scale with the measurement bar. (C) SRs 1, 3, 5, and 7, and 2, 4, 6, and 8 of nesprin-3α are shown aligned with the 13th and 14th SRs of spectrin-β2, respectively. Dark gray boxes depict alignment of more than three similar amino acids in an alignment set. Light gray boxes depict similar amino acids to a majority of those in the opposing alignment set. A diamond indicates that residues are conserved in only one alignment set, and an asterisk indicates residues that are conserved in both sets. Below the SR alignment the structures of nesprin-3α and -3β are shown. The location of the exons are denoted by bars and labeled in between each accordingly as E1–E18. The region of nesprin-3α encoded by the cDNA found in the Y2H screen is underlined, as is the sequence for nesprin-3β (RIKEN clone 4831426I19) found in the database. The SRs are depicted as open boxes. In exon 18, the RIKEN sequence continues for another 2,198 nucleotides after the stop codon. The NH2 terminus is to the left and the COOH terminus is to the right. The distances are drawn to scale.

Mentions: A basic local alignment search tool search with the novel plectin-1C ABD binding sequence showed stretches of nucleotide sequences identical with an Institute of Physical and Chemical Research (RIKEN) complementary DNA (cDNA; 4831426I19) and homology to a region in nesprin-1 and -2 containing a variable number of SRs. In the mouse genome, the nesprin-1 gene is on chromosome 10A1 (human homologue 6q25) and the nesprin-2 gene is on chromosome 12C3 (human homologue 14q23.2). Software analysis revealed that the gene for the novel nesprin-related sequence is also on chromosome 12, but in a different location than nesprin-2, in region 12E (human homologue 14q32.13, showing 72% homology). Database searches revealed that the cDNA for the nesprin-related protein sequence is also present in cDNA libraries of several other species, including the rat, chimpanzee, dog, and chicken (68, 68, 50, and 43% homology, respectively). Furthermore, the RIKEN clone contained a COOH-terminal KASH domain that was similar to that found in nesprin-1 and -2 (51 and 61% protein sequence homology, respectively; Fig. 2 A). These database analyses indicate that the nesprin-related cDNA sequence encodes a novel nesprin family member, which will be referred to as nesprin-3.


Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin.

Wilhelmsen K, Litjens SH, Kuikman I, Tshimbalanga N, Janssen H, van den Bout I, Raymond K, Sonnenberg A - J. Cell Biol. (2005)

Sequence analysis of nesprin-3. (A) Scaled diagrams of the nesprin-1, -2, -3α, and -3β, ANC-1, UNC-83, and D. melanogaster Klarsicht protein are presented. The sequence alignment of the KASH domain of the different murine nesprins, ANC-1 and UNC-83, with the conserved domain in the D. melanogaster Klarsicht protein is shown with similar amino acids shaded gray. (B) Organization of the nesprin-3 genome. The 18 exons are labeled as E1–E18 and are depicted as closed bars, except the exon unique to nesprin-3α (E3), which is depicted as an open box. The translation start sites for nesprin-3α and -3β are shown in E2. The distances are drawn to scale with the measurement bar. (C) SRs 1, 3, 5, and 7, and 2, 4, 6, and 8 of nesprin-3α are shown aligned with the 13th and 14th SRs of spectrin-β2, respectively. Dark gray boxes depict alignment of more than three similar amino acids in an alignment set. Light gray boxes depict similar amino acids to a majority of those in the opposing alignment set. A diamond indicates that residues are conserved in only one alignment set, and an asterisk indicates residues that are conserved in both sets. Below the SR alignment the structures of nesprin-3α and -3β are shown. The location of the exons are denoted by bars and labeled in between each accordingly as E1–E18. The region of nesprin-3α encoded by the cDNA found in the Y2H screen is underlined, as is the sequence for nesprin-3β (RIKEN clone 4831426I19) found in the database. The SRs are depicted as open boxes. In exon 18, the RIKEN sequence continues for another 2,198 nucleotides after the stop codon. The NH2 terminus is to the left and the COOH terminus is to the right. The distances are drawn to scale.
© Copyright Policy
Related In: Results  -  Collection

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fig2: Sequence analysis of nesprin-3. (A) Scaled diagrams of the nesprin-1, -2, -3α, and -3β, ANC-1, UNC-83, and D. melanogaster Klarsicht protein are presented. The sequence alignment of the KASH domain of the different murine nesprins, ANC-1 and UNC-83, with the conserved domain in the D. melanogaster Klarsicht protein is shown with similar amino acids shaded gray. (B) Organization of the nesprin-3 genome. The 18 exons are labeled as E1–E18 and are depicted as closed bars, except the exon unique to nesprin-3α (E3), which is depicted as an open box. The translation start sites for nesprin-3α and -3β are shown in E2. The distances are drawn to scale with the measurement bar. (C) SRs 1, 3, 5, and 7, and 2, 4, 6, and 8 of nesprin-3α are shown aligned with the 13th and 14th SRs of spectrin-β2, respectively. Dark gray boxes depict alignment of more than three similar amino acids in an alignment set. Light gray boxes depict similar amino acids to a majority of those in the opposing alignment set. A diamond indicates that residues are conserved in only one alignment set, and an asterisk indicates residues that are conserved in both sets. Below the SR alignment the structures of nesprin-3α and -3β are shown. The location of the exons are denoted by bars and labeled in between each accordingly as E1–E18. The region of nesprin-3α encoded by the cDNA found in the Y2H screen is underlined, as is the sequence for nesprin-3β (RIKEN clone 4831426I19) found in the database. The SRs are depicted as open boxes. In exon 18, the RIKEN sequence continues for another 2,198 nucleotides after the stop codon. The NH2 terminus is to the left and the COOH terminus is to the right. The distances are drawn to scale.
Mentions: A basic local alignment search tool search with the novel plectin-1C ABD binding sequence showed stretches of nucleotide sequences identical with an Institute of Physical and Chemical Research (RIKEN) complementary DNA (cDNA; 4831426I19) and homology to a region in nesprin-1 and -2 containing a variable number of SRs. In the mouse genome, the nesprin-1 gene is on chromosome 10A1 (human homologue 6q25) and the nesprin-2 gene is on chromosome 12C3 (human homologue 14q23.2). Software analysis revealed that the gene for the novel nesprin-related sequence is also on chromosome 12, but in a different location than nesprin-2, in region 12E (human homologue 14q32.13, showing 72% homology). Database searches revealed that the cDNA for the nesprin-related protein sequence is also present in cDNA libraries of several other species, including the rat, chimpanzee, dog, and chicken (68, 68, 50, and 43% homology, respectively). Furthermore, the RIKEN clone contained a COOH-terminal KASH domain that was similar to that found in nesprin-1 and -2 (51 and 61% protein sequence homology, respectively; Fig. 2 A). These database analyses indicate that the nesprin-related cDNA sequence encodes a novel nesprin family member, which will be referred to as nesprin-3.

Bottom Line: 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.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 alpha6beta4 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
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 alpha6beta4 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.

Show MeSH