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First published online February 23, 2005
doi: 10.1242/10.1242/jcs.01667

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Identification of trichoplein, a novel keratin filament-binding protein

Miwako Nishizawa^1,*, Ichiro Izawa^1,*, Akihito Inoko^1,*, Yuko Hayashi¹, Koh-ichi Nagata¹, Tomoya Yokoyama^1,2, Jiro Usukura³ and Masaki Inagaki^1,

¹ Division of Biochemistry, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan
² Department of Dermatology, Mie University Faculty of Medicine, 2-174 Edobashi, Tsu 514-8507, Japan
³ Department of Anatomy and Cell Biology, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan

View larger version (39K): [in a new window]   Fig. 1. Identification of trichoplein as a keratin-interacting protein. (A) Amino acid sequence of trichoplein from the full-length cDNA. The sequence contained in the original yeast clone is indicated with an arrow. (B) Scheme of the trichoplein protein with the trichohyalin/plectin homology domain (TPHD). Numbers refer to amino acid position.

View larger version (33K): [in a new window]   Fig. 2. In vitro interaction of trichoplein with keratin IFs. (A) Interactions of the C-terminal region of trichoplein (73-498 amino acids) or full-length trichoplein (1-498) with various IFs in the two-hybrid system. Y190 cells co-transformed with various pGBD-IFs and the pGAD-C-terminal region of trichoplein or pGAD-full-length trichoplein were selected in tryptophan- and leucine-free media and subjected to a ß-galactosidase filter assay. The numbers of plus signs represent the relative rates that the transformed yeast colonies turned blue after incubation at 30°C on filters: +++, <3 hours; ++, 3-8 hours; +, >8 hours. Minus signs represent colonies remaining white at 24 hours. Yeast cotransformed with pGBD-K8 and pGAD-K18, which started to turn blue in less than 3 hours (designated +++) on the filter assay, served as a positive control. (B) Interaction of endogenous K8/18 with GST-trichoplein (73-498 a.a.). The extracts of HeLa (lanes 1 and 2), T24 (lanes 3 and 4) and DJM-1 (lanes 5 and 6) cells were incubated with either GST (lanes 1, 3, and 5) or GST-trichoplein (lanes 2, 4, and 6) fixed on glutathione beads and proteins attached to the beads were detected using K8 or K18 antibody. (C) Co-sedimentation analyses of trichoplein with K8/18. K8/18 filaments were assembled on their own (lane 1), or in the presence of MBP (lane 2) or MBP-trichoplein (lane 4). As a control, only MBP-trichoplein (lane 3) was incubated in the assembly buffer. Vimentin (lane 5) and desmin (lane 6) filaments were assembled in the presence of MBP-trichoplein. Samples of supernatant (S) and pellet (P) fractions were separated by SDS-PAGE, and then stained with Coomassie Brilliant Blue (CBB) (top panel), or detected with MBP antibody by immunoblotting (WB, bottom panel). Molecular weight markers are shown on the left. The arrows indicate the position of MBP-trichoplein. (D) Co-sedimentation of trichoplein with polymerized tubulin and actin. MBP-trichoplein was incubated with (lane 2) or without (lane 1) polymerized actin (left panel) or polymerized tubulin (right panel). Samples of supernatant (S) and pellet (P) fractions were separated on SDS-PAGE, and stained with CBB. The arrow indicates the position of MBP-trichoplein.

View larger version (72K): [in a new window]   Fig. 3. Overexpressed GFP-tagged trichoplein is associated with K8/18 filaments in HeLa cells. (A) pEGFP-trichoplein vector was transfected into HeLa cells. The transfected cells were fixed 16 hours later and stained with K18 antibody, tubulin antibody or rhodamine-conjugated phalloidin. The merged images are also shown. (B) HeLa cells expressing GFP-trichoplein were treated with 10 ng/ml demecolcine (Dem.) for 100 minutes, or 5 µM cytochalasin B (Cyt.) for 15 minutes at 37°C. After the treatment, cells were fixed and stained with K18 antibody, tubulin antibody or rhodamine-conjugated phalloidin. (C) In some cells, GFP-trichoplein existed not only around nuclei but also in the cell periphery, where GFP-trichoplein markedly colocalized with K18. Higher magnification images (lower panels) of the areas indicated by arrows and merged images are also shown. Bar, 10 µm.

View larger version (97K): [in a new window]   Fig. 5. Colocalization of trichoplein and K8/18 filaments in HeLa cells. (A) HeLa cells were double-stained with trichoplein and K18 antibodies. The enlargement of the area indicated by an arrowhead and merged images are also shown. (B) Electron microscopic immunolocalization of trichoplein in HeLa cells. Antibody binding sites were detected predominantly in keratin-rich regions (left). At high magnification (right), staining is obvious on the bundle of keratin filaments (arrow). (C) HeLa cells in mitosis were double-stained for K18 and trichoplein. Bar, 10 µm (A,C); 500 nm (B, left); 50 nm (B, right).

View larger version (64K): [in a new window]   Fig. 4. In vivo association of trichoplein with K8/18. (A) Detection of trichoplein in HeLa, T24 and DJM-1 cells. Lysates from HeLa, T24 and DJM-1 cells were stained with CBB, detected with trichoplein antibody by immunoblotting (-trichoplein), or immunostained with trichoplein antibody preabsorbed by GST-trichoplein protein (absorbed). Trichoplein antibody specifically recognized bands of about 61 kDa in samples. Molecular size markers are shown on the left. (B) Interaction of trichoplein with K8/18 in vivo. Immunoprecipitates were prepared from lysates of HeLa, T24 and DJM-1 cells using control rabbit IgG and trichoplein antibody. Each precipitate was subjected to immunoblot analysis with trichoplein, K8 or K18 antibody.

View larger version (106K): [in a new window]   Fig. 6. Colocalization of trichoplein with K8/18 and desmoplakin in polarized Caco-2 cells. Caco-2 cells were cultured on laminin-coated cell culture inserts for more than 3 days. They were fixed and immunostained with antibodies as indicated and analyzed by laser-scanning confocal microscopy. Images a-f and j-o are in the X-Y plane. Images g-i are in the Z-section. (A) Caco-2 cells showed concentrations of K8/18 in the cortical cytoskeleton with higher density in apical region (a,d,g). At the upper and the middle level (a-i), trichoplein associates with K8/18 in a speckled pattern. Trichoplein also concentrated at the cell-cell border of the upper level (b). (B) Trichoplein colocalized with desmoplakin clearly at the cell-cell border of the upper level (j-l) and partially at the middle level (m-o). Bar, 10 µm.

View larger version (67K): [in a new window]   Fig. 7. Expression and distribution of trichoplein in various tissues. (A) Homogenates of various tissues from rats were immunoblotted with trichoplein antibody. Each lane contained 30 µg of protein from selected tissues. Trichoplein seemed to be expressed ubiquitously. (B) Double-staining of various tissues with trichoplein antibody and markers. In liver (a-c), trichoplein was exclusively concentrated and merged with K8/18 filaments in tubular structures (bile canaliculi). On the other hand, in brain (g,h) or skeletal muscle (i,j), trichoplein was not concentrated at NF or desmin-positive IFs. In addition, we found that the blood vessels are trichoplein-positive (d-f). (C) Vertical view (k) and horizontal view (l) of hair follicle stained with trichoplein antibody. Trichoplein localized at the outer root sheath (arrowheads in k,l), where K6/16 is found. (D) Double staining of the brain with trichoplein and PECAM-1 antibodies (m-o). Trichoplein localized to the blood vessels where PECAM-1 was also positive. Bar, 10 µm.

View larger version (95K): [in a new window]   Fig. 8. Distribution of trichoplein in the absorptive cells of the small intestine. Frozen sections of the small intestine were double stained with trichoplein antibody and the markers. Then, they were analyzed by laser-scanning confocal microscopy. (A) Double-stained absorptive cells with K8/18 and trichoplein antibodies. In the lower magnification of a nearly vertical view (a-c) and in the higher magnification of a horizontal view (d-f), trichoplein was associated with K8/18 in the apical region (terminal web). Panel e and arrowheads in b show trichoplein concentrating at the cell-cell border. (B) Double-stained absorptive cells with desmoplakin and trichoplein antibodies. In the lower magnification image of a nearly vertical view (g-i) and the higher magnification of a horizontal view (j-l), trichoplein is closely associated with desmoplakin at the cell-cell border. (C) Higher magnification of the vertical sectional views of the absorptive cells double stained with trichoplein antibody and K8/18 antibody or antibodies for the markers of junctional complex (ZO-1, ß-catenin or desmoplakin 1&2). Trichoplein is located close to K8/18-positive signals of higher density in the apical region (m-o). At the cell-cell border, trichoplein closely associates with desmoplakin (p-r), and does not always merge with ZO-1 (s-u) or ß-catenin (v-x). These observations are similar to those of Caco-2 cells in Fig. 6. Bar, 10 µm.

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