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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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Y2H analysis of the interaction between either the nesprin-related Y2H sequence or the first pair of FNIII domains of β4 with several different ABDs. Interactions were scored + when the plating efficiencies on selective SC-LTHA plates were >30% of those on nonselective SC-LT plates at 5 d of growth. Interactions were scored − when no colonies were detected at 10 d of growth. The percent homology of the other ABDs to the plectin ABD is shown in the column on the left. The results for the interactions of the first pair of FNIII domains with the ABDs listed have been reported previously (Litjens et al., 2003).
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fig1: Y2H analysis of the interaction between either the nesprin-related Y2H sequence or the first pair of FNIII domains of β4 with several different ABDs. Interactions were scored + when the plating efficiencies on selective SC-LTHA plates were >30% of those on nonselective SC-LT plates at 5 d of growth. Interactions were scored − when no colonies were detected at 10 d of growth. The percent homology of the other ABDs to the plectin ABD is shown in the column on the left. The results for the interactions of the first pair of FNIII domains with the ABDs listed have been reported previously (Litjens et al., 2003).

Mentions: It is well documented that the integrin β4 subunit is bound to plectin in hemidesmosomes (Borradori and Sonnenberg, 1999). We have shown previously that the critical binding site between plectin and β4 occurs through the first pair of fibronectin type III (FNIII) domains of β4 and the NH2-terminal ABD of plectin (Geerts et al., 1999; Litjens et al., 2003; Koster et al., 2004b). We were curious to find out if additional proteins could bind to the plectin ABD. For this purpose, we performed a Y2H genetic screen using the human plectin-1C ABD as bait. We identified several novel interactions with both known and previously unidentified proteins. Of the unknown proteins, one showed homology with the related type II ONM proteins, nesprin-1 and -2. We then tested the ability of the nesprin-related protein sequence to bind to other ABDs in a Y2H assay, using the previously published results for the first pair of FNIII domains of β4 to bind the same ABDs as a control (Litjens et al., 2003). The results show that, like β4, the nesprin-related sequence binds to the plectin-1C ABD and the BPAG1-n ABD, which is highly homologous to the plectin ABD (76% homology). However, the nesprin-related protein sequence, also like β4, did not bind to the ABDs of dystrophin, α-actinin-1, and filamin-A and -B (Fig. 1). We conclude that the nesprin-related sequence has the same binding specificity as the β4 subunit for the different ABDs.


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)

Y2H analysis of the interaction between either the nesprin-related Y2H sequence or the first pair of FNIII domains of β4 with several different ABDs. Interactions were scored + when the plating efficiencies on selective SC-LTHA plates were >30% of those on nonselective SC-LT plates at 5 d of growth. Interactions were scored − when no colonies were detected at 10 d of growth. The percent homology of the other ABDs to the plectin ABD is shown in the column on the left. The results for the interactions of the first pair of FNIII domains with the ABDs listed have been reported previously (Litjens et al., 2003).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171291&req=5

fig1: Y2H analysis of the interaction between either the nesprin-related Y2H sequence or the first pair of FNIII domains of β4 with several different ABDs. Interactions were scored + when the plating efficiencies on selective SC-LTHA plates were >30% of those on nonselective SC-LT plates at 5 d of growth. Interactions were scored − when no colonies were detected at 10 d of growth. The percent homology of the other ABDs to the plectin ABD is shown in the column on the left. The results for the interactions of the first pair of FNIII domains with the ABDs listed have been reported previously (Litjens et al., 2003).
Mentions: It is well documented that the integrin β4 subunit is bound to plectin in hemidesmosomes (Borradori and Sonnenberg, 1999). We have shown previously that the critical binding site between plectin and β4 occurs through the first pair of fibronectin type III (FNIII) domains of β4 and the NH2-terminal ABD of plectin (Geerts et al., 1999; Litjens et al., 2003; Koster et al., 2004b). We were curious to find out if additional proteins could bind to the plectin ABD. For this purpose, we performed a Y2H genetic screen using the human plectin-1C ABD as bait. We identified several novel interactions with both known and previously unidentified proteins. Of the unknown proteins, one showed homology with the related type II ONM proteins, nesprin-1 and -2. We then tested the ability of the nesprin-related protein sequence to bind to other ABDs in a Y2H assay, using the previously published results for the first pair of FNIII domains of β4 to bind the same ABDs as a control (Litjens et al., 2003). The results show that, like β4, the nesprin-related sequence binds to the plectin-1C ABD and the BPAG1-n ABD, which is highly homologous to the plectin ABD (76% homology). However, the nesprin-related protein sequence, also like β4, did not bind to the ABDs of dystrophin, α-actinin-1, and filamin-A and -B (Fig. 1). We conclude that the nesprin-related sequence has the same binding specificity as the β4 subunit for the different ABDs.

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