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First published online 13 May 2008
doi: 10.1242/jcs.019075

Journal of Cell Science 121, 1887-1898 (2008)
Published by The Company of Biologists 2008
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Nesprin-2 Giant (NUANCE) maintains nuclear envelope architecture and composition in skin

Yvonne Lüke1,*, Hafida Zaim1,*, Iakowos Karakesisoglou1,2,*, Verena M. Jaeger1, Lorenz Sellin3, Wenshu Lu1, Maria Schneider1, Sascha Neumann1,4, Asa Beijer1, Martina Munck1,4, V. C. Padmakumar1,4, Joachim Gloy3, Gerd Walz3 and Angelika A. Noegel1,4,{ddagger}

1 Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Cologne, Germany
2 Department of Biological Sciences, The School of Biological and Biomedical Sciences, The University of Durham, Durham DH1 3LE, UK
3 Renal Division, University Hospital Freiburg, 79106 Freiburg, Germany
4 Center for Molecular Medicine Cologne (CMMC) and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany


Figure 1
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  		Fig. 1. Generation of Nesprin-2-Giant KO mice. (A) Diagram illustrating the partial genomic organization of the Nesprin-2 (WT) locus coding for the ABD domain (yellow boxes), the targeting construct and the targeted allele (mutant allele). The Nesprin-2-Giant ATG translation start site in exon 1 is indicated with an arrow. The location of the 5' and 3' probes used for Southern blot analysis is also indicated. Primer sets correspond to those used in PCR analysis for identifying the WT (1.2 kb, Primer set 1: 5'-CATAGAACATGCCCTGACATTCCTG-3' and 5'-CTGTTTCTGAGTATTGCATGCGCTTG-3') and mutant (1.1 kb, Primer set 2: 5'-TGTCTGCCTACATGTTACTATGGTC-3' and 5'-TGCGAGGCCAGAGGCCACTTGTGTAGC-3') alleles. Primers used for RT-PCR are indicated (see Fig. 9 for more details). Ex, exon; E, EcoRI; S, SacI; K, KpnI. (B) Southern blot analysis of mouse-tail DNAs digested with EcoRI for 5' analysis and SacI for 3' analysis. (C) PCR analysis of DNAs isolated from Nesprin-2+/+, Nesprin-2+/– and Nesprin-2–/– mice. (D) Western blot analysis of HaCaT (human keratinocytes), and primary Nesprin-2+/+ and Nesprin-2–/– (clone 1 and 2) fibroblast homogenates. Blotting with pAbK1 indicates the absence of Nesprin-2 Giant in KO fibroblasts (arrowhead) and the presence of Nesprin-2 C-terminal isoforms (asterisks). For HaCaT cells, ECL signals were obtained after 5-minutes exposure; for dermal fibroblasts, signals were obtained after prolonged detection. (E) Structural features of all known Nesprin-2 isoforms. Major domains are indicated in different colours and shapes. The epitopes and identity of various anti-Nesprin-2 antibodies used are indicated (inverted Y).

 

Figure 2
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  		Fig. 2. Characterization of mAb K56-386. (A) Nesprin-2 pAbK1-immunoprecipitated PAM212 samples were probed with K56-386, which specifically recognizes Nesprin-2 Giant (arrowhead). (B) Equal amounts of HaCaT and PAM212 (mouse) cell lysates were probed with K56-386 and anti-β-tubulin (loading control). (C-G) K56-386 (1:10 dilution) stains the NE in PAM212 keratinocytes (C-E) and in WT mouse-skin frozen sections (F), and is largely absent in KO skin (G). Phalloidin staining delineates the plasma membrane. (G,H) K56-386 (1:10) staining is largely absent in KO epidermis (G); rarely, some residual NE staining is detectable in isolated regions in the KO epidermis, when K56-386 is used undiluted (inset in H). (I-K) The staining pattern of Nesprin-2 Giant is distinct from that of Nesprin-1 Giant in human (I,J) and mouse (K) epidermis. Nesprin-1-Giant localizes to the NE (I, arrowheads) and to cell-cell junctions (I, arrows). Insets, higher-magnifications of boxed areas. epi, epidermis; de, dermis; hf, hair follicle.

 

Figure 3
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  		Fig. 3. Generation of the Nesprin-2{Delta}ABD isoform in KO cells and tissues through alternative translational initiation. (A) The coding nucleotide sequences of Nesprin-2 exons 1-10 are shown and the deduced amino acid sequence is indicated beneath. Similar to other canonical CH1-CH2-type ABDs, the ABD of Nesprin-2 Giant is also encoded by seven exons (CH1-encoded protein sequence is coloured in yellow, linker region in white and CH2 in blue). Exons 2-4 (yellow) were deleted in Nesprin-2{Delta}ABD KO mice. The names and sequences of primers employed in the RT-PCR analysis are indicated in blue lettering. The K56-386 epitope in exon 9 is marked as a red box. (B) RT-PCR analysis of KO- and WT-fibroblast cDNAs using the primer sets indicated in A shows absence of the exon-2–exon-4 region (Ex2Fw/Ex4Rv set). RT-PCR employing exon-1- and exon-8-specific primers (Ex1Fw/Ex8Rv set) on WT- and KO-fibroblast RNA samples did not reveal the anticipated 130-bp exon-1&8 cDNA; instead, a 628-bp KO band was evident, compared with the expected 860-bp WT band (exons 1 through 8). Concomitant sequencing of the KO fragment indicated a splicing event of exon 1 with exon 5. The presence of these exons in the Nesprin-2{Delta}ABD transcript was verified by RT-PCR using the Ex5Fw/Ex7Rv set of primers. Because this alternative splicing event does not result in an open reading frame on the translated transcript, we searched the following exons for alternative initiation sites. Using the Chang Bioscience software (http://www.changbioscience.com/primo/ti.html), alternative KOZAK initiation-site sequences were identified in exons 7 and 8 [indicated in boldface (red) and overline in A]. (C) Semi-quantitative RT-PCR analysis of fibroblast cDNAs using the exon-1- and exon-8-specific primer set to show accumulation of the Nesprin-2{Delta}ABD transcript only in aged KO fibroblasts. WT analysis shows the expected 860-bp band (exon 1 through 8), whereas the KO displays the 628-bp band in fibroblasts of higher passages, indicating the alternative splicing event of exon 1 with exon 5. The number of passages is shown (#1, #3, #5).

 

Figure 4
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  		Fig. 4. Localization of the Nesprin-2 C-terminal isoform is affected in KO epidermis. (A-F) WT (A-C) and KO (D-F) frozen skin sections were stained with the N- and C-terminally directed anti-Nesprin-2 antibodies K56-386 and pAbK1. pAbK1 stains the NE in WT (B, arrowheads in inset), which is rarely apparent in the KO epidermis (E, arrowhead in inset). Hf, hair follicle; de, dermis; epi, epidermis. The broken line delineates the location of the basement membrane. Scale bars: 10 µm. (G) Nesprin-2-Giant–K56-386 immunocomplexes from mouse keratinocytes (PAM212) were probed (WB) with pAbK1 and K56-386. Arrowheads, small C-terminal Nesprin-2 isoforms; arrows, Nesprin-2-Giant isoform. Detection of the 800-kDa band in the K56-386 immunoblot is rarely possible in the input, but clearly visible in the immunoprecipitation lane. For the pAbK1 immunoblot, the sample was run in parallel on a separate gel. Silver staining is used as control (HC: IgG heavy chain).

 

Figure 5
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  		Fig. 5. Silencing of Nesprin-2 Giant in human keratinocytes affects nuclear shape and Nesprin-2 C-terminal-isoform localization. (A) Indirect immunofluorescence analysis of control and Nesprin-2-Giant-silenced HaCaT cells using Nesprin-2-Giant-specific antibodies (K20-478). Nuclei are stained by DAPI. Arrows indicate the NE phenotypes observed. Scale bar: 10 µm. (B) Histogram representing a statistical evaluation of specific NE phenotypes in control and Nesprin-2-Giant knockdown HaCaT cells (300 cells counted for each experiment). Error bars denote s.e.m. (C) Control and Nesprin-2-Giant knockdown cells were subjected to indirect immunofluorescence analysis using Nesprin-2-Giant-specific (K20-478) and Nesprin-2 polyclonal (pAbK1) antibodies; the latter also detects the C-terminal isoforms. Note that the pAbK1 pattern is affected or absent in Nesprin-2-Giant-silenced cells (asterisks). Nuclei are stained by DAPI. Scale bar: 10 µm. Confocal images are shown. (D) Immunoblot analysis of Nesprin-2-Giant-silenced human keratinocytes and control cells. Note the loss of the 800-kDa Giant isoform (arrowhead) in Nesprin-2-Giant-silenced human keratinocytes. Silencing of the Nesprin-2-Giant isoform also results in a downregulation of the smaller isoforms (*) detected by pAbK1. Actin is shown for control.

 

Figure 6
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  		Fig. 6. Nesprin-2-Giant KO mice are partial loss-of-function mutants. (A,B) pAbK1 immunoblot analysis of various WT and KO tissue homogenates (equal amounts loaded). Certain pAbK1-reactive Nesprin-2 C-terminal isoforms are present (arrows) and their levels are perturbed in specific KO tissues. Nesprin-2 Giant is absent in KO lysates. K56-386 detects Nesprin-2 Giant only in the WT (A). Emerin staining shows minor changes. (C,D) pAbK1 detected sarcomeric structures (arrows) and the NE (arrowheads) in WT (C) and KO (D) adult-mouse skeletal muscle frozen sections. DAPI, nuclei.

 

Figure 7
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  		Fig. 7. Epidermal proliferation and differentiation appears normal in KO epidermis. Skins from day-4 WT (A,C,E,G,I,K) and KO (B,D,F,H,J,L) mice were fixed, sectioned and stained with antibodies and with phalloidin to label F-actin. Colour codings correspond to the secondary antibodies or phalloidin used in each case. Note the increased KO epidermal thickness. Inset, higher-magnification of the boxed area. {alpha}6β4, {alpha}6β4 integrin; β1, β1 integrin; phall, phalloidin; Ker14, Keratin 14; Ker1, Keratin 1; Ker10, Keratin 10; E-Cad, E-cadherin; epi, epidermis; de, dermis; hf, hair follicle. Scale bars: 10 µm.

 

Figure 8
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  		Fig. 8. Nesprin-2 Giant affects nuclear morphology and size in the epidermis. (A-F) WT (A-C) and KO (D-F) frozen skin sections were stained with emerin-specific antibodies as indicated in the lower left of each frame. Note the increased nuclear size of KO epithelial cells. Emerin staining is NE-restricted in WT dermal cells (arrowheads, A-C), whereas, in the KO dermis (D,E and arrows in F), an abnormal staining pattern is evident. (G-L) Primary KO (G-I) and WT (J-L) keratinocytes were subjected to indirect immunofluorescence using anti-Nesprin-2 antibodies as indicated. K56-386-negative cells have an abnormal pAbK1 staining and misshapen nuclei (arrows, H,I). Cells exhibiting weak K56-386 staining (asterisks, G) have a normal nucleus and have pAbK1 staining at the NE (arrowheads, H). Note that the K56-386 and pAbK1 stainings are restricted to the NE in WT cells (K). Insets in J-L are higher-magnifications from another area of the same cover slip. Insets in A,D,I are higher-magnifications of the respective boxed areas in B,E,I, respectively. Confocal images are shown. epi, epidermis; de, dermis; hf, hair follicle. Scale bars: 10 µm.

 

Figure 9
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  		Fig. 9. Nuclear architecture and emerin localization is affected in Nesprin-2-Giant KO fibroblasts. In WT cells (A,B,E,F), Nesprin-2 Giant (A) and C-terminal isoforms (pAbK1; E, arrows) are present at the NE, whereas primary KO cells (C,D,G,H) are negative for Nesprin-2 Giant (C), display strong pAbK1 staining in the cytoplasm (arrows, G) and exhibit severely misshapen nuclei (arrowheads, D,H). Representative nuclear changes are shown in I-L. Note that a pAbK1 NE staining in KO cells coincides with a normal nuclear architecture (asterisks, G,H). (M-O) In higher-passage (>5) KO cells, the mutant nuclear defects diminish (N,O), presumably due to the weak expression of an abnormally spliced {Delta}CH1Nesprin-2-Giant protein (M). (O) Histogram representing a statistical evaluation of control and mutant cells (percentage of cells; >700 cells counted for each cell type) displaying nuclear deformations. The passage cell number is indicated at the middle of each individual histogram. Error bars denote s.d. Significant P value for control versus early-passage mutants = 4x10–5 (Student's t-test). (P-S) Immunostainings, employing emerin-specific antibodies, of WT (P) and KO (Q-S) cells. In contrast to WT (P), emerin is unevenly localized at misshapen NEs in KO cells (arrowheads, Q). (S) Representative examples are shown in S. Note the emerin accumulation and clustering in the deformed NE regions (arrowheads). Confocal images are shown.

 

Figure 10
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  		Fig. 10. Loss of Nesprin-2 Giant affects fibroblast migration and cell polarity. (A-F) In contrast to WT fibroblasts (A-C), wounds generated by scratching remain unclosed in Nesprin-2-Giant-deficient fibroblasts (D-F). Fibroblasts of passage 2 were used and KO fibroblasts were verified to be negative for K56-382 staining prior to usage. A representative experiment is shown from three separate experiments. Scale bars: 200 µm. (G) Mutant cells exhibit a statistically significant reduced migration velocity. Mean values and s.d. were calculated from three different primary WT and five different mutant cell cultures, which were assayed in triplicate experiments. (H,I) Indirect immunofluorescence examination of WT and Nesprin-2-Giant-deficient cell monolayers 6-hours post-wounding, using antibodies against Golgi (GM130; row H) and MTOC ({gamma}-tubulin; row I). The broken lines indicate the migrating cell front, which was visualized by FITC-phalloidin counterstaining. Note that both the Golgi complex and MTOC are not positioned towards the wound edge in the mutants. (H',I') Statistical evaluation of the representative experiments shown in H,I indicates a defective cell polarization in Nesprin-2-Giant mutants. Cells with Golgi structures (500 cells counted for each cell type) and MTOCs (300 cells counted for each cell type) positioned within a 120° sector facing the wound edge were assessed as polarized. Error bars denote s.d. from three different experiments. The statistical significance (P values; Student's t-test) is also indicated. Scale bar: 10 µm.

 

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© The Company of Biologists Ltd 2008