Myosin and Actin in Hair Follicle 115 Fig. 1. Immunoblot of skin proteins reacted with antibodies against myosin and actin. A) Rat dorsal skin proteins (lanes 1, 3) or mystacial pad proteins (lanes 2, 4), obtained from the animals at postpartum day 7, were reacted with mouse monoclonal antibody (clone A4) against human skeletal muscle myosin heavy chain (lanes 1, 2) or were reacted with rabbit polyclonal antibody (BT561) against human platelet myosin heavy chain (lane 3, 4). B) Rat dorsal skin proteins (lane 1) or mystacial pad proteins (lane 2), obtained from the animals at postpartum day 7, were reacted with mouse monoclonal antibody (clone C4) against chicken gizzard actin. Lanes A5 and B3 represent the molecular weight markers (rabbit skeletal muscle myosin, 223 kDa; E. coli -galactosidase, 116 kDa; bovine serum albumin, 66 kDa; rabbit skeletal muscle aldolase, 42 kDa; bovine erythrocyte carbonic anhydrase, 30 kDa; horse skeletal muscle myoglobin, 17 kDa). Localization of myosin by peroxidase method Hematoxylin-eosin staining and immunohistochemical visualization of the myosin were carried out for the sections obtained from dorsal and mystacial skin tissues. Although the epidermal structures of the ordinary hair follicle and that of the vibrissal follicle are essentially the same, the dermal tissues surrounding both types of follicles are very different [5]. The vibrissal follicle is surrounded by a highly devel- oped sinus and capsule in the intermediate height area (Fig. 2C). These dermal tissues are reduced at the upper height area of the follicle (Fig. 2D). In the upper area, the hair shaft was not stained by hematoxylin-eosin (Fig. 2D). Generally, rat skin consists of epidermis, dermal con- nective tissue, and dermal muscle, arranged from outside to inside, as shown in Figure 2A and E. This order is disrupted in the mystacial pad, where the dermal muscle invades the dermal connective tissue area up to just beneath the epider- mis, as detected by the antibody against skeletal muscle myosin (Fig. 2F and G) in addition to the histological view (Fig. 2B). The vibrissal follicle is surrounded by a skeletal muscle (not arrector pili), which is reserved for the voluntary movement of the whiskers (Fig. 2H). Although cells of the outer root sheath in Figure 2H are faintly stained, we have not determined whether or not this is due to the cross-reac- tivity of the antibody. Generally other vibrissal follicle cells were not stained for skeletal myosin (Fig. 2H and I). In the inserted Figure 2J in Figure 2I as well as Figure 3A, B rep- resents control experiments without the addition of primary antibodies. Non-specific staining was not observed except that at the bottom end of the epidermal cornified layer (Fig. 3A). Figure 3C shows that the nonmuscle myosin II is located in the spinous (arrow b) and granular (arrow c) layers of the epidermis, while it appears to be scarce in the proliferative basal layer (arrow a). These results suggest that myosin II may participate in forming a stratified struc- ture of the epidermis as a component of the cellular mem- brane cortex and/or cytoskeleton, which probably provides sealing and strength to the skin. The signal of myosin II appears to be eliminated when the cells enter into the kera- tinization stage (Fig. 3C and D). Compared with the hair follicle cells, both interfollicu- lar dermal cells and epidermal cells give a strong signal of myosin II (Fig. 3D). Nevertheless, some cells in the hair follicle were stained. One group of these stained cells was observed in the hair bulb, as indicated by the thick arrows in Fig. 3E and the arrowheads in Fig. 3G. These cells were gen- erally adjacent to the dermal papillae. The other group of cells is located around the middle height of the outer root sheath (Fig. 3F, thick arrow; 3G, thin arrow; and 3H, thin arrow ORS). In addition to these groups, the peripheral area (corresponding to the hair cuticle) of the hair shaft may be stained specifically (Fig. 3H, arrow HS). This is further investigated by immunofluorescence staining and the result was confirmed as shown in Figure 7; the details are men- tioned in a latter part of this paper. The companion layer was not necessarily stained as shown by the arrow Cp in Figure 3H. Nonmuscle myosin II was distributed widely in both epidermal and dermal tissues, including skeletal muscle [15, 21, 27] (Fig. 3). It is interesting to note that most of the basal layer of skin epidermis, hair matrix, dermal papillae, hair cortex, and inner root sheath exhibited a very low signal level for nonmuscle myosin II.