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First published online 15 February 2005
doi: 10.1242/jcs.01683

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Uni-axial stretching regulates intracellular localization of Hic-5 expressed in smooth-muscle cells in vivo

¹ Department of Microbiology, Showa University School of Pharmaceutical Sciences, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
² Department of Oral Pathology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
³ Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

View larger version (92K): [in a new window]   Fig. 1. Distribution of Hic-5 in adult mouse tissues. (A) Immunoreactivity with anti-Hic-5 monoclonal antibody (middle), anti--SM-actin monoclonal antibody (bottom) and control IgG (top) in the aorta (left) and the large intestine (right). (B) The colocalization of endogenous and transfected Hic-5. The cells were transfected with the expression vector of HA-tagged Hic-5 (pCG-LD1mhic-5) subjected to uni-axial cyclic stretch at 1 Hz for 60 minutes (stretch +) or not (stretch –) and immunostained with both anti-HA (Y-11) antibody and anti-Hic-5 antibody. The anti-Hic-5 antibody stained the same types of structures in the transfected as in the neighboring untransfected cells, supporting the colocalization of the endogenous and exogenous proteins. The arrow indicates the direction of stretch. Bars, 5 µm.

View larger version (78K): [in a new window]   Fig. 2. Translocation of Hic-5 from focal adhesions to actin stress fibers during uni-axial cyclic stretch. The Tet-Off cell lines of Hic-5 (A) and paxillin (B) were incubated without Dox for 24 hours, subjected to uni-axial cyclic stretching at 1 Hz for 60 minutes (+) or not (–) and stained with the antibody 12CA5 against HA-tag of Hic-5 (A, top) and paxillin (B, top). The cells were also stained with FITC-conjugated phalloidin (middle) and merged images of phalloidin and HA tag of Hic-5 or paxillin are shown on the right. SVS30 SM cells were subjected to uni-axial cyclic stretching at 1 Hz for 60 minutes (+) or not (–), and stained with the antibody against Hic-5 and with FITC-conjugated phalloidin (C) and with the antibody against paxillin and FITC-conjugated phalloidin (D). The arrow indicates the direction of stretching. Bars, 10 µm.

View larger version (68K): [in a new window]   Fig. 3. Intracellular distribution of Hic-5-binding proteins during uni-axial cyclic stretch. Mouse embryo fibroblast cells subjected to the uni-axial cyclic stretch for 60 minutes (+) or not (–), as in Fig. 2, were stained with antibodies against endogenous vinculin (A, top) and GIT1 (D, top). (B,C) The expression vector encoding myc-tagged FAK (B) or HA-tagged PTP-PEST (C) was introduced into mouse embryo fibroblast cells and, at 24 hours after transfection, the cells were subjected to cyclic stretching as above and stained with antibodies against tags [9E10 (B) and 12CA5 (C)]. The distribution of actin stress fibers was visualized by FITC-conjugated phalloidin labeling (A-D, middle). The arrow indicates the direction of stretching. Bars, 10 µm.

View larger version (72K): [in a new window]   Fig. 4. Intracellular distribution of CRP2 and -actinin during uni-axial cyclic stretch. Mouse embryo fibroblast cells were transfected with Flag-tagged CRP2 expression vector, subjected to cyclic stretching (+) or not (–) and stained with the polyclonal antibody against the Flag tag (A) or with that against endogenous -actinin (B). The insets in the merged images are magnification of a part of a cell's rectangular frame. The white arrows in the inset of B show a periodic pattern of -actinin staining. Bars, 10 µm. (C) The Hic-5 domains interacting with the factors and presence (+) or absence (–) of the factors on stress fiber during the cyclic stretching.

View larger version (65K): [in a new window]   Fig. 5. The C-terminal region of Hic-5 is sufficient for the stress-fiber-restricted localization under cyclic stretching. (A) The HA-tagged Hic-5 deletion mutants used in this experiment. (B) The expression plasmids of the Hic-5 mutants were transfected into mouse embryo fibroblast cells; 24 hours later, western blotting was carried out using the antibody to the HA tag. (C) Mouse embryo fibroblast cells were transfected with each expression vector, subjected to cyclic stretching (+) or not (–) and immunostained with the 12CA5 antibody against the HA tag (left, red). (C, middle) F-actin stained by FITC-conjugated phalloidin (green); (C, right) merged images. The arrow indicates the direction of stretching. Bars, 5 µm.

View larger version (74K): [in a new window]   Fig. 6. The LIM2 and LIM3 domains are crucial for Hic-5 to be localized to stress fibers. (A) The HA-tagged Hic-5 mutants in which each of the LIMs was disrupted by point mutations (mL1-mL3) or a deletion (delL4). The expression plasmids for each mutant have been previously described (Shibanuma et al., 2003). (B) The expression plasmids were transfected into mouse embryo fibroblast cells; 24 hours later, western blotting was carried out using the antibody against the HA tag. (C) Mouse embryo fibroblast cells were transfected with each expression vector, subjected to cyclic stretching (+) or not (–) and immunostained with the 12CA5 antibody against the HA tag. (C, left) Staining of the Hic-5 mutants (red) with the 12CA5 antibody to the HA tag; (C, middle) F-actin stained by FITC-conjugated phalloidin (green); (C, right) merged images. The arrow indicates the direction of stretch. Bars, 5 µm.

View larger version (81K): [in a new window]   Fig. 7. Hic-5 associates and localizes with CRP2 to actin stress fibers. (A) Flag-tagged CRP2 was expressed with HA-tagged Hic-5 wild-type and mutants in COS7 cells: lane 1, wild-type; lane 2, N-paxillin/C-Hic; lane 3, N-Hic/C-paxillin; lane 4, paxillin. 24 hours after transfection, the cells were lysed and subjected to immunoprecipitation with polyclonal anti-HA antibody (lane H) or normal rabbit IgG (lane C) as a control, followed by immunoblotting with monoclonal anti-HA (top) or anti-Flag (M2) (bottom) antibodies. (B) The wild-type and chimeric proteins of Hic-5 and paxillin studied in A. (C) Hic-5 (pCG-LD1mhic-5) and CRP2 (Flag-CRP2) co-expressed in mouse embryo fibroblast cells were co-immunostained using the monoclonal anti-HA (12CA5) and polyclonal anti-Flag antibodies after being exposed to the cyclic stretching. The top, middle and bottom images show Hic-5, CRP2 and a merged image, respectively. The arrow indicates the direction of stretching. Bar, 2 µm. (D) Immunogold electron microscopy of a mouse aorta. (top) L, lumen; ET, endothelial cell; EL, elastic lamina; SMC, smooth-muscle cell. (bottom) Magnified image of a part of the smooth-muscle cell (framed rectangle at top). Arrows indicate immunogold (10 nm) labeling of Hic-5 and arrowheads indicate immunogold (15 nm) labeling of CRP2. (middle) A similar field exposed to control serum. Bars, 2 µm (top) and 100 nm (middle and bottom).

View larger version (39K): [in a new window]   Fig. 8. Effects of Hic-5, paxillin and CRP2 on collagen-gel contraction. (A) The Tet-Off/LD1mhic-5 (top) and the MEF/Tet-Off/paxillin (bottom) cell lines incubated with (Dox +) or without (Dox –) doxycycline for 24 hours were embedded in collagen gels and the gel contraction was assessed by measuring the diameters at each indicated time. The means and standard deviations of four parallel measurements are shown. (B, top) The Tet-Off/LD1mhic-5 cell line incubated with (Dox +) or without (Dox –) doxycycline for 24 hours was infected with the retrovirus of CRP2 (CRP +) or control virus (CRP –) for the next 24 hours and then embedded in collagen gel. The gel contraction was assessed as in A and the pictures were taken 56 hours after floating the collagen gels (C). (B, bottom) Expression plasmids encoding the mL2 and mL3 Hic-5 mutants were introduced into SVS30 smooth-muscle cells by electroporation and, 24 hours later, the cells were embedded in collagen gels and observed as in A. (D) Intracellular distribution of Hic-5 and CRP2 in the cells embedded in collagen gel. The cells in collagen gel were prepared as in C and stained with anti-Hic-5 (red) and anti-Flag polyclonal (CRP2) (green) antibodies, and DAPI (blue). Bar, 10 µm.

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