Limits...
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.

Show MeSH
Endogenous nesprin-3 and plectin associate in TM-4 cells. (A) The mouse Sertoli cell line TM-4 was transiently transfected with either the pSUPER vector expressing the nesprin-3 siRNA (A–D) or the luciferase siRNA (E–H), along with an expression vector for mRFP at a 5:1 ratio. After 72 h, the cells were fixed and stained for endogenous plectin (A and E) and nesprin-3 using our pAbs (C and G). mRFP was visualized to determine which cells express the siRNAs (B and F). D and H are composite images of the first three images in each row. All images are maximum projections. Bar, 20 μM. (B) TM-4 cells were left untransfected (A–H) or transiently transfected with the pSUPER vector expressing the nesprin-3 siRNA, along with an expression vector for mRFP in a 5:1 ratio (I–L). After 72 h and just before fixation, the cells in images E–L were treated for 30 min with 0.2 μM latrunculin B. The cells were then fixed and stained for plectin (B, F, and J), nesprin-3 (C, G, and K), and F-actin (A and E). mRFP was visualized to determine which cells express the nesprin-3 siRNA (I). D, H, and L are composite images of the first three images in each row. Bar, 10 μM.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171291&req=5

fig6: Endogenous nesprin-3 and plectin associate in TM-4 cells. (A) The mouse Sertoli cell line TM-4 was transiently transfected with either the pSUPER vector expressing the nesprin-3 siRNA (A–D) or the luciferase siRNA (E–H), along with an expression vector for mRFP at a 5:1 ratio. After 72 h, the cells were fixed and stained for endogenous plectin (A and E) and nesprin-3 using our pAbs (C and G). mRFP was visualized to determine which cells express the siRNAs (B and F). D and H are composite images of the first three images in each row. All images are maximum projections. Bar, 20 μM. (B) TM-4 cells were left untransfected (A–H) or transiently transfected with the pSUPER vector expressing the nesprin-3 siRNA, along with an expression vector for mRFP in a 5:1 ratio (I–L). After 72 h and just before fixation, the cells in images E–L were treated for 30 min with 0.2 μM latrunculin B. The cells were then fixed and stained for plectin (B, F, and J), nesprin-3 (C, G, and K), and F-actin (A and E). mRFP was visualized to determine which cells express the nesprin-3 siRNA (I). D, H, and L are composite images of the first three images in each row. Bar, 10 μM.

Mentions: We next wanted to verify that the nesprin-3 siRNA can knock-down the expression of the protein in TM-4 cells and to determine the location of plectin in these cells. The nesprin-3 siRNA– or luciferase control siRNA–containing vectors were independently transfected into TM-4 cells, along with a monomeric red fluorescent protein (mRFP) expression plasmid, and the cells were stained for endogenous plectin and nesprin-3 (Fig. 6 A). The mRFP protein was visualized to determine whether the cells express the siRNAs. The results show that the nesprin-3 siRNA can specifically knock-down the expression of the protein at the NE in TM-4 cells. The results also show that the knock-down of nesprin-3 does not obviously affect the distribution of plectin, but do show that in some cells plectin is concentrated around the NE as reported previously (Guttman et al., 1999). Interestingly, in fully spread cells plectin is primarily present in FCs and colocalized with F-actin (Fig. 6 B). Like that from MEF cells, the data obtained from TM-4 cells suggests that nesprin-3α and actin filaments compete for binding to plectin. Therefore, we decided to disrupt the actin network in TM-4 cells using latrunculin B. This should have resulted in an increased amount of plectin available to interact with nesprin-3α at the ONM and, indeed, this is what was observed (Fig. 6 B). To determine if the recruitment of plectin to the nuclear perimeter is dependent on the presence of nesprin-3α, we expressed the nesprin-3 siRNA–containing vector in TM-4 cells and treated the cells with latrunculin B. The concentration of plectin around the nuclear perimeter in these cells was significantly reduced, with plectin now being distributed in the cytoplasm (Fig. 6 B), which suggests that the recruitment of plectin to the ONM after actin depolymerization is indeed dependent on nesprin-3α. These data also infer that the plectin that is concentrated around the nuclear perimeter in cells that were not fully spread is also dependent on an association with nesprin-3α. Together, the data presented thus far clearly demonstrate that nesprin-3α can bind plectin at the ONM in cells.


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)

Endogenous nesprin-3 and plectin associate in TM-4 cells. (A) The mouse Sertoli cell line TM-4 was transiently transfected with either the pSUPER vector expressing the nesprin-3 siRNA (A–D) or the luciferase siRNA (E–H), along with an expression vector for mRFP at a 5:1 ratio. After 72 h, the cells were fixed and stained for endogenous plectin (A and E) and nesprin-3 using our pAbs (C and G). mRFP was visualized to determine which cells express the siRNAs (B and F). D and H are composite images of the first three images in each row. All images are maximum projections. Bar, 20 μM. (B) TM-4 cells were left untransfected (A–H) or transiently transfected with the pSUPER vector expressing the nesprin-3 siRNA, along with an expression vector for mRFP in a 5:1 ratio (I–L). After 72 h and just before fixation, the cells in images E–L were treated for 30 min with 0.2 μM latrunculin B. The cells were then fixed and stained for plectin (B, F, and J), nesprin-3 (C, G, and K), and F-actin (A and E). mRFP was visualized to determine which cells express the nesprin-3 siRNA (I). D, H, and L are composite images of the first three images in each row. Bar, 10 μM.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Endogenous nesprin-3 and plectin associate in TM-4 cells. (A) The mouse Sertoli cell line TM-4 was transiently transfected with either the pSUPER vector expressing the nesprin-3 siRNA (A–D) or the luciferase siRNA (E–H), along with an expression vector for mRFP at a 5:1 ratio. After 72 h, the cells were fixed and stained for endogenous plectin (A and E) and nesprin-3 using our pAbs (C and G). mRFP was visualized to determine which cells express the siRNAs (B and F). D and H are composite images of the first three images in each row. All images are maximum projections. Bar, 20 μM. (B) TM-4 cells were left untransfected (A–H) or transiently transfected with the pSUPER vector expressing the nesprin-3 siRNA, along with an expression vector for mRFP in a 5:1 ratio (I–L). After 72 h and just before fixation, the cells in images E–L were treated for 30 min with 0.2 μM latrunculin B. The cells were then fixed and stained for plectin (B, F, and J), nesprin-3 (C, G, and K), and F-actin (A and E). mRFP was visualized to determine which cells express the nesprin-3 siRNA (I). D, H, and L are composite images of the first three images in each row. Bar, 10 μM.
Mentions: We next wanted to verify that the nesprin-3 siRNA can knock-down the expression of the protein in TM-4 cells and to determine the location of plectin in these cells. The nesprin-3 siRNA– or luciferase control siRNA–containing vectors were independently transfected into TM-4 cells, along with a monomeric red fluorescent protein (mRFP) expression plasmid, and the cells were stained for endogenous plectin and nesprin-3 (Fig. 6 A). The mRFP protein was visualized to determine whether the cells express the siRNAs. The results show that the nesprin-3 siRNA can specifically knock-down the expression of the protein at the NE in TM-4 cells. The results also show that the knock-down of nesprin-3 does not obviously affect the distribution of plectin, but do show that in some cells plectin is concentrated around the NE as reported previously (Guttman et al., 1999). Interestingly, in fully spread cells plectin is primarily present in FCs and colocalized with F-actin (Fig. 6 B). Like that from MEF cells, the data obtained from TM-4 cells suggests that nesprin-3α and actin filaments compete for binding to plectin. Therefore, we decided to disrupt the actin network in TM-4 cells using latrunculin B. This should have resulted in an increased amount of plectin available to interact with nesprin-3α at the ONM and, indeed, this is what was observed (Fig. 6 B). To determine if the recruitment of plectin to the nuclear perimeter is dependent on the presence of nesprin-3α, we expressed the nesprin-3 siRNA–containing vector in TM-4 cells and treated the cells with latrunculin B. The concentration of plectin around the nuclear perimeter in these cells was significantly reduced, with plectin now being distributed in the cytoplasm (Fig. 6 B), which suggests that the recruitment of plectin to the ONM after actin depolymerization is indeed dependent on nesprin-3α. These data also infer that the plectin that is concentrated around the nuclear perimeter in cells that were not fully spread is also dependent on an association with nesprin-3α. Together, the data presented thus far clearly demonstrate that nesprin-3α can bind plectin at the ONM in cells.

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