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doi: 10.1242/10.1242/jcs.00191

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Interaction of periplakin and envoplakin with intermediate filaments

¹ Keratinocyte Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
² Department of Dermatology, Kurume University School of Medicine, 67 Asahimachi, Kurume, Fukuoka 830, Japan

View larger version (47K): [in a new window]   Fig. 1. The C-terminus of periplakin. (A) Alignment of the periplakin C-terminus with the linker sequences of other plakins. Light blue, identical amino acids; dark blue, conserved amino acids. Human periplakin (PPL) is shown from amino acid 1646; human envoplakin (EVPL) from amino acid 1675; human desmoplakin (DP) from amino acid 2454, rat plectin from amino acid 3722 and human BPAG1 from amino acid 2327 (Mahoney et al., 1998; Määttä et al., 2000). (B) Amino acid sequence of periplakin C-terminus, with residues that are most highly conserved between plakins (i.e. light or dark blue in A) shaded. The assignment of the boundary between the rod domain and linker domain is that described by Määttä et al. and DiColandrea et al. (Määttä et al., 2000; DiColandrea et al., 2000), except that the numbering has been corrected by one amino acid. (C) Periplakin C-terminal constructs tested in immunofluorescence and overlay assays. P, periplakin; R, rod domain; L, linker domain. The association of each construct with intermediate filaments (IF) was evaluated by immunofluorescence in transiently transfected COS7 cells stained with (+extraction) or without (-extraction) prior extraction and in overlay assays (overlay) with keratinocyte and COS7 cell intermediate filament preparations. +, colocalisation that was sufficiently strong to make the distribution of periplakin almost indistinguishable from intermediate filaments (strong binding in overlay assays); +/-, partial colocalisation in which the filamentous distribution of the periplakin constructs was evident without the need to overlay the intermediate filament staining pattern (weak binding in overlay assays); -, no obvious filamentous distribution; n.d., not determined.

View larger version (103K): [in a new window]   Fig. 2. Effects of detergent extraction on COS7 cells. (A-C) Scanning electron microscopy of cells extracted in 0.6% saponin for 15 minutes (A) or 30 minutes (B,C). Area boxed in B is shown at higher magnification in C. (D-F) Immunofluorescence staining of cells fixed without prior extraction (D) or cells extracted with saponin for 30 minutes (E,F). Green fluorescence: anti-ß tubulin (D,E); anti-vimentin (F). Red fluorescence: phalloidin (D-F). Purple fluorescence: nuclei stained with TOTO-3. Bars, 10 µm (A,B,D-F), 1 µm (C).

View larger version (163K): [in a new window]   Fig. 3. Transfection of COS7 cells and keratinocytes with periplakin C-terminal constructs. (A-L) COS7 cells; (M-P) primary human epidermal keratinocytes. After transfection, the cells were fixed without prior extraction (A,E-G,I,M-P) or extracted with 0.6% saponin for 30 minutes before fixation (B-D,H,J-L). B and C are the same field; J-L are the same field; M and N are the same field; and O and P are the same field. See Fig. 1 for details of each construct. Green fluorescence: anti-HA (detects tagged periplakin constructs). Red fluorescence: anti-vimentin in COS7 cells; anti-keratin (LP34) in keratinocytes. Purple/blue fluorescence: nuclei stained with TOTO-3, Bars, 10 µm (A-C,E,F,I); 50 µm (D,G,H); 5 µm (J-P).

View larger version (28K): [in a new window]   Fig. 4. Overlay assays of the interaction of periplakin constructs with intermediate filaments. (A) Autoradiography of [35S]-methionine-labeled in vitro translated probes. Mobility of molecular mass standards of 200, 150, 100, 75, 50, 37 and 25 kDa are indicated. (B) Binding of [35S]-methionine-labeled periplakin recombinant proteins to unlabelled intermediate filaments (IF) extracted from cultured epidermal keratinocytes and COS7 cells. CBB: Coomassie Brilliant Blue staining of intermediate filament preparations prior to transfer to nitrocellulose. Probes are described in Figs 1 and 5 or as follows. P-R, periplakin rod domain; pCI-neo, empty vector control; DP-C, desmoplakin construct with intact C-terminus [DPNwt (Stappenbeck and Green, 1992)]; K5/K14, keratin 5 and 14.

View larger version (15K): [in a new window]   Fig. 5. Summary of constructs with rod domain deletions. P, periplakin; E, envoplakin; full, full length protein; N, N-terminus; R, rod domain; L, linker domain; C, C box subdomain of C terminus; a.a., amino acid.

View larger version (54K): [in a new window]   Fig. 6. Immunofluorescence analysis of COS7 cells transfected with full length envoplakin (E-full) or periplakin (P-full) or constructs lacking the rod domains (envoplakin, ERod; peiplakin, PRod). The cells were fixed without prior extraction (A-C,E,F,H,J-L) or extracted before fixation (D,G,I). Green fluorescence: anti-FLAG (detects tagged envoplakin; A,C-E,G-J,L); anti-HA (detects tagged periplakin; B). Red fluorescence: anti-HA (C,F,G,H,K,L); anti-vimentin (A,B,D,I). Blue fluorescence: nuclei stained with TOTO-3. In D note residual nuclear fluorescence attributable to ERod. Bars, 20 µm (C-H); 5 µm (A,B,I-L).

View larger version (116K): [in a new window]   Fig. 7. Immunofluorescence analysis of COS7 cells transfected with E-1/2N1/2R alone (A-H) or in combination with P-full (K,L) or with P-full alone (I,J). Cells were fixed without prior extraction (A-D, I-K) or extracted with 0.6% saponin before fixation (E-H,L). Green fluorescence: anti-FLAG (to detect tagged envoplakin; A,B,D-F,H,K,L); anti-HA (to detect tagged periplakin; I,J). Red fluorescence: anti-vimentin (A,C-E,G-I,K,L), anti-keratin (J). Boxed areas in A,E are shown at higher magnification in B-D and F-H, respectively. Bars, 10 µm (A-H,L); 5 µm (I-K).

View larger version (125K): [in a new window]   Fig. 8. Immunofluorescence analysis of COS7 cells transfected with envoplakin C-terminal constructs, alone (A-H) or in combination with the periplakin P-1/4R+L construct (I-L). Cells were fixed without prior extraction (A-C,E-G,I) or extracted before fixation (D,H,J-L). Green fluorescence: anti-FLAG (to detect tagged envoplakin). Red fluorescence: anti-vimentin (A,C,D,E,G,H); anti-HA (to detect tagged periplakin; I,K,L). Blue/purple fluorescence: nuclei stained with TOTO-3. Boxed inserts in A,E,I are shown at higher magnification in A-C, E-G and I, respectively. J-L are the same field. In D there is no residual green fluorescence and therefore no evidence that the cells shown were actually transfected. In H the central cell has weak residual green fluorescence. Bars, 10 µm.

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