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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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Tissue and cell line distribution of nesprin-3. (A) The indicated mouse-derived cell lines were lysed with RIPA buffer to extract NE components. Whole cell lysates (WCLs) of untransfected, nesprin-3α cDNA–transfected, or nesprin-3β cDNA–transfected COS7 cells were included as controls. WCL samples were loaded and run in a 4–20% SDS-PAGE gel, transferred to PVDF membrane, and probed for the presence of nesprin-3 using the GST-nesprin-3-SR7–derived antibodies. A protein blot for β-actin was used as a loading control. (B) The indicated mouse tissues were dounce homogenized in RIPA buffer. WCLs of these samples were analyzed as in B. A protein blot for α-tubulin was used as a loading control. (C) To determine the selectivity of nesprin-3α and -3β expression, the mouse cell lines NMK-1, mTSC, and mBCC-1 were used. Primers annealing on either side of the nesprin-3α–specific exon 3 (primers 1 and 2) were used to determine the presence of either variant in these cell lines (top; see Results). Primers that would amplify exons 15–18 of nesprin-3 (primers 3 and 4) were used as a control for mRNA fidelity and specificity (middle). Nesprin-3α and -3β cDNAs were used as controls in these PCR reactions. A PCR of GAPDH was also included as a control for nesprin-3 expression levels and mRNA fidelity (bottom). Schematic representations (Fig. 2 C) of the regions of nesprin-3α and -3β amplified by primer pairs are shown underneath the image. Only part of nesprin-3 is depicted, for simplicity.
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fig3: Tissue and cell line distribution of nesprin-3. (A) The indicated mouse-derived cell lines were lysed with RIPA buffer to extract NE components. Whole cell lysates (WCLs) of untransfected, nesprin-3α cDNA–transfected, or nesprin-3β cDNA–transfected COS7 cells were included as controls. WCL samples were loaded and run in a 4–20% SDS-PAGE gel, transferred to PVDF membrane, and probed for the presence of nesprin-3 using the GST-nesprin-3-SR7–derived antibodies. A protein blot for β-actin was used as a loading control. (B) The indicated mouse tissues were dounce homogenized in RIPA buffer. WCLs of these samples were analyzed as in B. A protein blot for α-tubulin was used as a loading control. (C) To determine the selectivity of nesprin-3α and -3β expression, the mouse cell lines NMK-1, mTSC, and mBCC-1 were used. Primers annealing on either side of the nesprin-3α–specific exon 3 (primers 1 and 2) were used to determine the presence of either variant in these cell lines (top; see Results). Primers that would amplify exons 15–18 of nesprin-3 (primers 3 and 4) were used as a control for mRNA fidelity and specificity (middle). Nesprin-3α and -3β cDNAs were used as controls in these PCR reactions. A PCR of GAPDH was also included as a control for nesprin-3 expression levels and mRNA fidelity (bottom). Schematic representations (Fig. 2 C) of the regions of nesprin-3α and -3β amplified by primer pairs are shown underneath the image. Only part of nesprin-3 is depicted, for simplicity.

Mentions: Comparison of the RIKEN cDNA 4831426I19 and the nesprin-3 Y2H nucleotide sequences with the mouse genomic sequence on chromosome 12 revealed that these two ORFs represent splice variants of the nesprin-3 gene because we identified alternatively spliced exon 1 sequences for the Y2H and RIKEN sequences, labeled exons 1a and 1b, respectively, and an additional 173-nucleotide exon in the Y2H sequence (exon 3; Fig. 2 B and Fig. S1, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200506083/DC1). Additionally, both sequences contain an identical exon 2, but the reading frame is different in this exon for the two nesprin-3 splice variants; therefore, these proteins have different NH2-terminal sequences (Fig. S1 A). RT-PCR analysis on total mRNA from several cell lines suggests that after exon 3 the nucleotide (and protein) sequence is the same in the two splice variants and is encoded by exons 4–18 (Fig. 2 B, Fig. 3 C, and Fig. S1 A). Because the nesprin-3 Y2H sequence was the originally discovered sequence and may represent a longer nesprin-3 variant, we will now refer to this variant as nesprin-3α, whereas the RIKEN cDNA–encoded polypeptide will be referred to as nesprin-3β.


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)

Tissue and cell line distribution of nesprin-3. (A) The indicated mouse-derived cell lines were lysed with RIPA buffer to extract NE components. Whole cell lysates (WCLs) of untransfected, nesprin-3α cDNA–transfected, or nesprin-3β cDNA–transfected COS7 cells were included as controls. WCL samples were loaded and run in a 4–20% SDS-PAGE gel, transferred to PVDF membrane, and probed for the presence of nesprin-3 using the GST-nesprin-3-SR7–derived antibodies. A protein blot for β-actin was used as a loading control. (B) The indicated mouse tissues were dounce homogenized in RIPA buffer. WCLs of these samples were analyzed as in B. A protein blot for α-tubulin was used as a loading control. (C) To determine the selectivity of nesprin-3α and -3β expression, the mouse cell lines NMK-1, mTSC, and mBCC-1 were used. Primers annealing on either side of the nesprin-3α–specific exon 3 (primers 1 and 2) were used to determine the presence of either variant in these cell lines (top; see Results). Primers that would amplify exons 15–18 of nesprin-3 (primers 3 and 4) were used as a control for mRNA fidelity and specificity (middle). Nesprin-3α and -3β cDNAs were used as controls in these PCR reactions. A PCR of GAPDH was also included as a control for nesprin-3 expression levels and mRNA fidelity (bottom). Schematic representations (Fig. 2 C) of the regions of nesprin-3α and -3β amplified by primer pairs are shown underneath the image. Only part of nesprin-3 is depicted, for simplicity.
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fig3: Tissue and cell line distribution of nesprin-3. (A) The indicated mouse-derived cell lines were lysed with RIPA buffer to extract NE components. Whole cell lysates (WCLs) of untransfected, nesprin-3α cDNA–transfected, or nesprin-3β cDNA–transfected COS7 cells were included as controls. WCL samples were loaded and run in a 4–20% SDS-PAGE gel, transferred to PVDF membrane, and probed for the presence of nesprin-3 using the GST-nesprin-3-SR7–derived antibodies. A protein blot for β-actin was used as a loading control. (B) The indicated mouse tissues were dounce homogenized in RIPA buffer. WCLs of these samples were analyzed as in B. A protein blot for α-tubulin was used as a loading control. (C) To determine the selectivity of nesprin-3α and -3β expression, the mouse cell lines NMK-1, mTSC, and mBCC-1 were used. Primers annealing on either side of the nesprin-3α–specific exon 3 (primers 1 and 2) were used to determine the presence of either variant in these cell lines (top; see Results). Primers that would amplify exons 15–18 of nesprin-3 (primers 3 and 4) were used as a control for mRNA fidelity and specificity (middle). Nesprin-3α and -3β cDNAs were used as controls in these PCR reactions. A PCR of GAPDH was also included as a control for nesprin-3 expression levels and mRNA fidelity (bottom). Schematic representations (Fig. 2 C) of the regions of nesprin-3α and -3β amplified by primer pairs are shown underneath the image. Only part of nesprin-3 is depicted, for simplicity.
Mentions: Comparison of the RIKEN cDNA 4831426I19 and the nesprin-3 Y2H nucleotide sequences with the mouse genomic sequence on chromosome 12 revealed that these two ORFs represent splice variants of the nesprin-3 gene because we identified alternatively spliced exon 1 sequences for the Y2H and RIKEN sequences, labeled exons 1a and 1b, respectively, and an additional 173-nucleotide exon in the Y2H sequence (exon 3; Fig. 2 B and Fig. S1, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200506083/DC1). Additionally, both sequences contain an identical exon 2, but the reading frame is different in this exon for the two nesprin-3 splice variants; therefore, these proteins have different NH2-terminal sequences (Fig. S1 A). RT-PCR analysis on total mRNA from several cell lines suggests that after exon 3 the nucleotide (and protein) sequence is the same in the two splice variants and is encoded by exons 4–18 (Fig. 2 B, Fig. 3 C, and Fig. S1 A). Because the nesprin-3 Y2H sequence was the originally discovered sequence and may represent a longer nesprin-3 variant, we will now refer to this variant as nesprin-3α, whereas the RIKEN cDNA–encoded polypeptide will be referred to as nesprin-3β.

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