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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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Nesprin-3α associates with plectin in cells. (A) COS7 cells were transiently transfected with either the nesprin-3α (lanes 1–5) or -3β (lanes 6–10) cDNA constructs, or a control plasmid (lanes 10–15) and expression constructs for the HA-plectin-1A ABD (lanes 1, 6, and 11), HA-plectin-1C ABD (lanes 2, 7, and 12), HA-MACF ABD (lanes 3, 8, and 13), HA-α-actinin-1 ABD (lanes 4, 9, and 14), or a control plasmid (lanes 5, 10, and 15). The cells were lysed in RIPA buffer and HA-IPs were probed for the presence of nesprin-3 (top) and the HA-tagged ABDs (middle). WCLs were probed for expression levels of the two different nesprin-3 isoforms (bottom). (B) MEF cells were stained for endogenous plectin using the mAb 121 (A) and nesprin-3 using rabbit pAbs (D). The location of the nucleus was visualized using Topro staining (B and E). C is a composite of A and B, and F is a composite of D and E. Bar, 20 μM. (C) PA-JEB (A–T) or PA-JEB/β4 (U–X) were stably transduced with retroviral constructs expressing either GFP-nesprin-3α (A–D, I–L, and Q–X) or GFP-nesprin-3β (E–H and M–P). Fluorescence studies were done to locate the GFP moiety (A, E, I, M, Q, and U), along with immunofluorescence studies of endogenous plectin (B, F, J, N, R, and V) and keratin-6 (C and G) or keratin-14 (K, O, S, and W). The far right image of each row depicts the composite image of the first three images in each row (D, H, L, P, T, and X). Four cells are shown in each image except for images Q–T, which show a higher magnification of the nucleus. All images are maximum projections. Bars: (A–P and U–X) 20 μM; (Q–T) 10 μM. (D) To determine the subcellular localization of nesprin-3 and plectin at the NE, ultrathin sections of PA-JEB cells stably expressing nesprin-3α were labeled with rabbit pAbs against the plectin-ABD (D16), followed by incubation with 10-nm gold-conjugated protein A. The sections were then fixed in 1% glutaraldehyde and reprobed with rabbit pAbs against GFP, followed by incubation with 15-nm gold-conjugated protein A. Arrows indicate areas where plectin and GFP-nesprin-3α are colocalized at the ONM (bottom). Control labeling was done in the same way, except that after plectin labeling and fixation in glutaraldehyde, the sections were reprobed with 15-nm gold-conjugated protein A only (top). Note that no labeling with 15-nm gold was seen at the NE. (E) PA-JEB/β4 cells stably expressing either GFP-nesprin-3α (A–F) or GFP-nesprin-3β (G–I) were visualized for GFP (A, D, and G) and plectin (B, E, and H). The far right image of each row depicts the composite image of the first two images in each row (C, F, and I). All images are maximum projections. Bar, 20 μM.
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fig5: Nesprin-3α associates with plectin in cells. (A) COS7 cells were transiently transfected with either the nesprin-3α (lanes 1–5) or -3β (lanes 6–10) cDNA constructs, or a control plasmid (lanes 10–15) and expression constructs for the HA-plectin-1A ABD (lanes 1, 6, and 11), HA-plectin-1C ABD (lanes 2, 7, and 12), HA-MACF ABD (lanes 3, 8, and 13), HA-α-actinin-1 ABD (lanes 4, 9, and 14), or a control plasmid (lanes 5, 10, and 15). The cells were lysed in RIPA buffer and HA-IPs were probed for the presence of nesprin-3 (top) and the HA-tagged ABDs (middle). WCLs were probed for expression levels of the two different nesprin-3 isoforms (bottom). (B) MEF cells were stained for endogenous plectin using the mAb 121 (A) and nesprin-3 using rabbit pAbs (D). The location of the nucleus was visualized using Topro staining (B and E). C is a composite of A and B, and F is a composite of D and E. Bar, 20 μM. (C) PA-JEB (A–T) or PA-JEB/β4 (U–X) were stably transduced with retroviral constructs expressing either GFP-nesprin-3α (A–D, I–L, and Q–X) or GFP-nesprin-3β (E–H and M–P). Fluorescence studies were done to locate the GFP moiety (A, E, I, M, Q, and U), along with immunofluorescence studies of endogenous plectin (B, F, J, N, R, and V) and keratin-6 (C and G) or keratin-14 (K, O, S, and W). The far right image of each row depicts the composite image of the first three images in each row (D, H, L, P, T, and X). Four cells are shown in each image except for images Q–T, which show a higher magnification of the nucleus. All images are maximum projections. Bars: (A–P and U–X) 20 μM; (Q–T) 10 μM. (D) To determine the subcellular localization of nesprin-3 and plectin at the NE, ultrathin sections of PA-JEB cells stably expressing nesprin-3α were labeled with rabbit pAbs against the plectin-ABD (D16), followed by incubation with 10-nm gold-conjugated protein A. The sections were then fixed in 1% glutaraldehyde and reprobed with rabbit pAbs against GFP, followed by incubation with 15-nm gold-conjugated protein A. Arrows indicate areas where plectin and GFP-nesprin-3α are colocalized at the ONM (bottom). Control labeling was done in the same way, except that after plectin labeling and fixation in glutaraldehyde, the sections were reprobed with 15-nm gold-conjugated protein A only (top). Note that no labeling with 15-nm gold was seen at the NE. (E) PA-JEB/β4 cells stably expressing either GFP-nesprin-3α (A–F) or GFP-nesprin-3β (G–I) were visualized for GFP (A, D, and G) and plectin (B, E, and H). The far right image of each row depicts the composite image of the first two images in each row (C, F, and I). All images are maximum projections. Bar, 20 μM.

Mentions: To determine the subcellular location of nesprin-3, we transiently transfected pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes with constructs expressing GFP- or VSV-tagged nesprin-3α and -3β proteins. The results indicate that GFP-nesprin-3α and -3β are localized at the NE, although there is some staining within the nucleus, which is likely caused by the folding of the NE. The location of nesprin-3 was confirmed by colocalization studies with endogenous lamin A, which is a structural protein known to reside at the nucleoplasmic side of the INM (Fig. 4 A). Immunofluorescence studies of the VSV-tagged nesprin-3α and -3β proteins and endogenous nesprin-3 also showed localization at the NE and, in some cells, at the RER (Fig. 5 B and not depicted). Together, these results definitively show that nesprin-3 is a NE component. The results also indicate that the GFP and VSV tags do not affect the localization of the nesprin-3 proteins. Because confocal microscopy is not sensitive enough to determine which lipid bilayer of the NE contains nesprin-3, we used EM analysis on PA-JEB cells stably expressing either GFP-nesprin-3α or -3β. Using antibodies directed against the GFP moiety, we were able to show that, like nesprin-1 and -2, nesprin-3 is also present in the ONM (Fig. 4 B; Starr and Han, 2002; Zhen et al., 2002). Furthermore, some cells showed strong staining of nesprin-3 in the RER (Fig. 4 B), which was always correlated with a higher level of nesprin-3 expression in individual cells (Fig. 5 B).


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)

Nesprin-3α associates with plectin in cells. (A) COS7 cells were transiently transfected with either the nesprin-3α (lanes 1–5) or -3β (lanes 6–10) cDNA constructs, or a control plasmid (lanes 10–15) and expression constructs for the HA-plectin-1A ABD (lanes 1, 6, and 11), HA-plectin-1C ABD (lanes 2, 7, and 12), HA-MACF ABD (lanes 3, 8, and 13), HA-α-actinin-1 ABD (lanes 4, 9, and 14), or a control plasmid (lanes 5, 10, and 15). The cells were lysed in RIPA buffer and HA-IPs were probed for the presence of nesprin-3 (top) and the HA-tagged ABDs (middle). WCLs were probed for expression levels of the two different nesprin-3 isoforms (bottom). (B) MEF cells were stained for endogenous plectin using the mAb 121 (A) and nesprin-3 using rabbit pAbs (D). The location of the nucleus was visualized using Topro staining (B and E). C is a composite of A and B, and F is a composite of D and E. Bar, 20 μM. (C) PA-JEB (A–T) or PA-JEB/β4 (U–X) were stably transduced with retroviral constructs expressing either GFP-nesprin-3α (A–D, I–L, and Q–X) or GFP-nesprin-3β (E–H and M–P). Fluorescence studies were done to locate the GFP moiety (A, E, I, M, Q, and U), along with immunofluorescence studies of endogenous plectin (B, F, J, N, R, and V) and keratin-6 (C and G) or keratin-14 (K, O, S, and W). The far right image of each row depicts the composite image of the first three images in each row (D, H, L, P, T, and X). Four cells are shown in each image except for images Q–T, which show a higher magnification of the nucleus. All images are maximum projections. Bars: (A–P and U–X) 20 μM; (Q–T) 10 μM. (D) To determine the subcellular localization of nesprin-3 and plectin at the NE, ultrathin sections of PA-JEB cells stably expressing nesprin-3α were labeled with rabbit pAbs against the plectin-ABD (D16), followed by incubation with 10-nm gold-conjugated protein A. The sections were then fixed in 1% glutaraldehyde and reprobed with rabbit pAbs against GFP, followed by incubation with 15-nm gold-conjugated protein A. Arrows indicate areas where plectin and GFP-nesprin-3α are colocalized at the ONM (bottom). Control labeling was done in the same way, except that after plectin labeling and fixation in glutaraldehyde, the sections were reprobed with 15-nm gold-conjugated protein A only (top). Note that no labeling with 15-nm gold was seen at the NE. (E) PA-JEB/β4 cells stably expressing either GFP-nesprin-3α (A–F) or GFP-nesprin-3β (G–I) were visualized for GFP (A, D, and G) and plectin (B, E, and H). The far right image of each row depicts the composite image of the first two images in each row (C, F, and I). All images are maximum projections. Bar, 20 μM.
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fig5: Nesprin-3α associates with plectin in cells. (A) COS7 cells were transiently transfected with either the nesprin-3α (lanes 1–5) or -3β (lanes 6–10) cDNA constructs, or a control plasmid (lanes 10–15) and expression constructs for the HA-plectin-1A ABD (lanes 1, 6, and 11), HA-plectin-1C ABD (lanes 2, 7, and 12), HA-MACF ABD (lanes 3, 8, and 13), HA-α-actinin-1 ABD (lanes 4, 9, and 14), or a control plasmid (lanes 5, 10, and 15). The cells were lysed in RIPA buffer and HA-IPs were probed for the presence of nesprin-3 (top) and the HA-tagged ABDs (middle). WCLs were probed for expression levels of the two different nesprin-3 isoforms (bottom). (B) MEF cells were stained for endogenous plectin using the mAb 121 (A) and nesprin-3 using rabbit pAbs (D). The location of the nucleus was visualized using Topro staining (B and E). C is a composite of A and B, and F is a composite of D and E. Bar, 20 μM. (C) PA-JEB (A–T) or PA-JEB/β4 (U–X) were stably transduced with retroviral constructs expressing either GFP-nesprin-3α (A–D, I–L, and Q–X) or GFP-nesprin-3β (E–H and M–P). Fluorescence studies were done to locate the GFP moiety (A, E, I, M, Q, and U), along with immunofluorescence studies of endogenous plectin (B, F, J, N, R, and V) and keratin-6 (C and G) or keratin-14 (K, O, S, and W). The far right image of each row depicts the composite image of the first three images in each row (D, H, L, P, T, and X). Four cells are shown in each image except for images Q–T, which show a higher magnification of the nucleus. All images are maximum projections. Bars: (A–P and U–X) 20 μM; (Q–T) 10 μM. (D) To determine the subcellular localization of nesprin-3 and plectin at the NE, ultrathin sections of PA-JEB cells stably expressing nesprin-3α were labeled with rabbit pAbs against the plectin-ABD (D16), followed by incubation with 10-nm gold-conjugated protein A. The sections were then fixed in 1% glutaraldehyde and reprobed with rabbit pAbs against GFP, followed by incubation with 15-nm gold-conjugated protein A. Arrows indicate areas where plectin and GFP-nesprin-3α are colocalized at the ONM (bottom). Control labeling was done in the same way, except that after plectin labeling and fixation in glutaraldehyde, the sections were reprobed with 15-nm gold-conjugated protein A only (top). Note that no labeling with 15-nm gold was seen at the NE. (E) PA-JEB/β4 cells stably expressing either GFP-nesprin-3α (A–F) or GFP-nesprin-3β (G–I) were visualized for GFP (A, D, and G) and plectin (B, E, and H). The far right image of each row depicts the composite image of the first two images in each row (C, F, and I). All images are maximum projections. Bar, 20 μM.
Mentions: To determine the subcellular location of nesprin-3, we transiently transfected pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes with constructs expressing GFP- or VSV-tagged nesprin-3α and -3β proteins. The results indicate that GFP-nesprin-3α and -3β are localized at the NE, although there is some staining within the nucleus, which is likely caused by the folding of the NE. The location of nesprin-3 was confirmed by colocalization studies with endogenous lamin A, which is a structural protein known to reside at the nucleoplasmic side of the INM (Fig. 4 A). Immunofluorescence studies of the VSV-tagged nesprin-3α and -3β proteins and endogenous nesprin-3 also showed localization at the NE and, in some cells, at the RER (Fig. 5 B and not depicted). Together, these results definitively show that nesprin-3 is a NE component. The results also indicate that the GFP and VSV tags do not affect the localization of the nesprin-3 proteins. Because confocal microscopy is not sensitive enough to determine which lipid bilayer of the NE contains nesprin-3, we used EM analysis on PA-JEB cells stably expressing either GFP-nesprin-3α or -3β. Using antibodies directed against the GFP moiety, we were able to show that, like nesprin-1 and -2, nesprin-3 is also present in the ONM (Fig. 4 B; Starr and Han, 2002; Zhen et al., 2002). Furthermore, some cells showed strong staining of nesprin-3 in the RER (Fig. 4 B), which was always correlated with a higher level of nesprin-3 expression in individual cells (Fig. 5 B).

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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Related in: MedlinePlus