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The nuclear envelope protein Nesprin-2 has roles in cell proliferation and differentiation during wound healing.

Rashmi RN, Eckes B, Glöckner G, Groth M, Neumann S, Gloy J, Sellin L, Walz G, Schneider M, Karakesisoglou I, Eichinger L, Noegel AA - Nucleus (2012)

Bottom Line: When we probed for an interaction of Nesprin-2 Giant with chromatin we observed in ChIP Seq experiments an association of the protein with heterochromatic and centromeric DNA.Through this activity Nesprin-2 can affect the nuclear landscape and gene regulation.Our findings suggest functions for Nesprin-2 at the nuclear envelope (NE) in gene regulation and in regulation of the actin cytoskeleton which impact on wound healing.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry I, Medical Faculty, University of Cologne, Cologne, Germany.

ABSTRACT
Nesprin-2, a type II transmembrane protein of the nuclear envelope, is a component of the LINC complex that connects the nuclear lamina with the actin cytoskeleton. To elucidate its physiological role we studied wound healing in Nesprin-2 Giant deficient mice and found that a loss of the protein affected wound healing particularly at later stages during fibroblast differentiation and keratinocyte proliferation leading to delayed wound closure. We identified altered expression and localization of transcription factors as one of the underlying mechanisms. Furthermore, the actin cytoskeleton which surrounds the nucleus was altered and keratinocyte migration was slowed down and focal adhesion formation enhanced. We also uncovered a new activity of Nesprin-2. When we probed for an interaction of Nesprin-2 Giant with chromatin we observed in ChIP Seq experiments an association of the protein with heterochromatic and centromeric DNA. Through this activity Nesprin-2 can affect the nuclear landscape and gene regulation. Our findings suggest functions for Nesprin-2 at the nuclear envelope (NE) in gene regulation and in regulation of the actin cytoskeleton which impact on wound healing.

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Migration and focal adhesion formation is altered in Nesprin-2G deficient keratinocytes. (A) A scratch-wound healing assay reveals a migration defect in Nesprin-2G deficient primary keratinocytes. Wound closure was followed by live cell microscopy (0 to 48 h after scratching). Scale bar, 200 µm. (B) The speed of migration was determined at the indicated time points (µm/hr). (C) Formation of focal adhesions in control and Nesprin-2G deficient primary keratinocytes. Keratinocytes were trypsinized and plated onto type I collagen. Focal adhesion formation was assessed after 3.5 and 6.5 h by staining for Vinculin. The boxed area shows focal contacts (arrows). Scale bar, 25 μm. (D) Determination of the percentage of cells that had formed focal adhesions 3.5 and 6.5 h after plating. 285 WT cells were analyzed each at both time points and 100 (3.5 h) and 130 (6.5 h) KO cells. (E) Determination of the area containing cells positive for Vinculin. 111 WT and 60 KO keratinocytes were examined. (F) protein gel blot analysis showing the expression of Vinculin protein in Nesprin-2 KD and WT cells. Actin was used as a loading control.
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Figure 5: Migration and focal adhesion formation is altered in Nesprin-2G deficient keratinocytes. (A) A scratch-wound healing assay reveals a migration defect in Nesprin-2G deficient primary keratinocytes. Wound closure was followed by live cell microscopy (0 to 48 h after scratching). Scale bar, 200 µm. (B) The speed of migration was determined at the indicated time points (µm/hr). (C) Formation of focal adhesions in control and Nesprin-2G deficient primary keratinocytes. Keratinocytes were trypsinized and plated onto type I collagen. Focal adhesion formation was assessed after 3.5 and 6.5 h by staining for Vinculin. The boxed area shows focal contacts (arrows). Scale bar, 25 μm. (D) Determination of the percentage of cells that had formed focal adhesions 3.5 and 6.5 h after plating. 285 WT cells were analyzed each at both time points and 100 (3.5 h) and 130 (6.5 h) KO cells. (E) Determination of the area containing cells positive for Vinculin. 111 WT and 60 KO keratinocytes were examined. (F) protein gel blot analysis showing the expression of Vinculin protein in Nesprin-2 KD and WT cells. Actin was used as a loading control.

Mentions: To ask whether altered keratinocyte migration contributes to the wound healing defects, we analyzed the migratory behavior of keratinocytes after introduction of scratch wounds into monolayers (Fig. 5A). Whereas WT keratinocytes had significantly migrated toward the scratch after 48 h, the open wound area in the KO was wider. Quantification of the speed of migration supported this difference revealing a slower migration for KO keratinocytes [WT, 4.27 ± 1.33, KO, 1,66 ± 0.48 μm/h (12 h); WT, 3,20 ± 0.53, KO, 0.91 ± 0.29 (24 h) and WT, 2.92 ± 0.38, KO, 0.69 ± 0.10 (48 h)] (Fig. 5B). Pictures taken at different time points using time lapse video microscopy confirmed these observations.


The nuclear envelope protein Nesprin-2 has roles in cell proliferation and differentiation during wound healing.

Rashmi RN, Eckes B, Glöckner G, Groth M, Neumann S, Gloy J, Sellin L, Walz G, Schneider M, Karakesisoglou I, Eichinger L, Noegel AA - Nucleus (2012)

Migration and focal adhesion formation is altered in Nesprin-2G deficient keratinocytes. (A) A scratch-wound healing assay reveals a migration defect in Nesprin-2G deficient primary keratinocytes. Wound closure was followed by live cell microscopy (0 to 48 h after scratching). Scale bar, 200 µm. (B) The speed of migration was determined at the indicated time points (µm/hr). (C) Formation of focal adhesions in control and Nesprin-2G deficient primary keratinocytes. Keratinocytes were trypsinized and plated onto type I collagen. Focal adhesion formation was assessed after 3.5 and 6.5 h by staining for Vinculin. The boxed area shows focal contacts (arrows). Scale bar, 25 μm. (D) Determination of the percentage of cells that had formed focal adhesions 3.5 and 6.5 h after plating. 285 WT cells were analyzed each at both time points and 100 (3.5 h) and 130 (6.5 h) KO cells. (E) Determination of the area containing cells positive for Vinculin. 111 WT and 60 KO keratinocytes were examined. (F) protein gel blot analysis showing the expression of Vinculin protein in Nesprin-2 KD and WT cells. Actin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3383573&req=5

Figure 5: Migration and focal adhesion formation is altered in Nesprin-2G deficient keratinocytes. (A) A scratch-wound healing assay reveals a migration defect in Nesprin-2G deficient primary keratinocytes. Wound closure was followed by live cell microscopy (0 to 48 h after scratching). Scale bar, 200 µm. (B) The speed of migration was determined at the indicated time points (µm/hr). (C) Formation of focal adhesions in control and Nesprin-2G deficient primary keratinocytes. Keratinocytes were trypsinized and plated onto type I collagen. Focal adhesion formation was assessed after 3.5 and 6.5 h by staining for Vinculin. The boxed area shows focal contacts (arrows). Scale bar, 25 μm. (D) Determination of the percentage of cells that had formed focal adhesions 3.5 and 6.5 h after plating. 285 WT cells were analyzed each at both time points and 100 (3.5 h) and 130 (6.5 h) KO cells. (E) Determination of the area containing cells positive for Vinculin. 111 WT and 60 KO keratinocytes were examined. (F) protein gel blot analysis showing the expression of Vinculin protein in Nesprin-2 KD and WT cells. Actin was used as a loading control.
Mentions: To ask whether altered keratinocyte migration contributes to the wound healing defects, we analyzed the migratory behavior of keratinocytes after introduction of scratch wounds into monolayers (Fig. 5A). Whereas WT keratinocytes had significantly migrated toward the scratch after 48 h, the open wound area in the KO was wider. Quantification of the speed of migration supported this difference revealing a slower migration for KO keratinocytes [WT, 4.27 ± 1.33, KO, 1,66 ± 0.48 μm/h (12 h); WT, 3,20 ± 0.53, KO, 0.91 ± 0.29 (24 h) and WT, 2.92 ± 0.38, KO, 0.69 ± 0.10 (48 h)] (Fig. 5B). Pictures taken at different time points using time lapse video microscopy confirmed these observations.

Bottom Line: When we probed for an interaction of Nesprin-2 Giant with chromatin we observed in ChIP Seq experiments an association of the protein with heterochromatic and centromeric DNA.Through this activity Nesprin-2 can affect the nuclear landscape and gene regulation.Our findings suggest functions for Nesprin-2 at the nuclear envelope (NE) in gene regulation and in regulation of the actin cytoskeleton which impact on wound healing.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry I, Medical Faculty, University of Cologne, Cologne, Germany.

ABSTRACT
Nesprin-2, a type II transmembrane protein of the nuclear envelope, is a component of the LINC complex that connects the nuclear lamina with the actin cytoskeleton. To elucidate its physiological role we studied wound healing in Nesprin-2 Giant deficient mice and found that a loss of the protein affected wound healing particularly at later stages during fibroblast differentiation and keratinocyte proliferation leading to delayed wound closure. We identified altered expression and localization of transcription factors as one of the underlying mechanisms. Furthermore, the actin cytoskeleton which surrounds the nucleus was altered and keratinocyte migration was slowed down and focal adhesion formation enhanced. We also uncovered a new activity of Nesprin-2. When we probed for an interaction of Nesprin-2 Giant with chromatin we observed in ChIP Seq experiments an association of the protein with heterochromatic and centromeric DNA. Through this activity Nesprin-2 can affect the nuclear landscape and gene regulation. Our findings suggest functions for Nesprin-2 at the nuclear envelope (NE) in gene regulation and in regulation of the actin cytoskeleton which impact on wound healing.

Show MeSH
Related in: MedlinePlus