Myosin and Actin in Hair Follicle 119 its significance is not known. The staining of the basal layer of epidermis (Fig. 4A, arrow a) was faint, whereas the spinous and granular layers were stained distinctly, as in the reaction with the anti-nonmuscle myosin antibody (Fig. 3C). Small arrows and arrowheads in Fig. 4B designate the stain- ing of the hair shaft. The infundibular area of the hair follicle is surrounded by a sheath connected to the basal layer of the epidermis. This monolayer sheath (or extended basal layer, Fig. 5A, asterisk) was clearly distinguishable from the outer root sheath, because the former is highly sensitive to hematoxy- lin staining. The peroxidase staining of this area by both anti-myosin and anti-actin antibodies was indistinct as compared with that of the outer root sheath (Fig. 5B and C, compare the areas of * and ORS) and was surrounded by an intercellular space corresponding to the basement membrane (Fig. 5A and B, BM). For both antibodies, the hair shaft and inner root sheath were not stained (Fig. 5B and C), though they were slightly reactive in lower areas (Fig. 5H and I). In the lower areas, endothelial cells, sinus, and capsule envelop the follicle (Fig. 5D and G) and the extended basal layer becomes indistinguishable in the lowest section (Fig. 5G). The capsule appears to be stained significantly (Fig. 5E and F). Colocalization of myosin and actin by immunofluorescence method The cells in the hair matrix stained with anti-nonmuscle myosin antibody (red) were shown to be identical to those stained with anti-actin antibody (green) as shown in Figure 6. Arrows in C and G denote the special cells rich in both nonmuscle myosin and actin (yellow) by overlapping Figure 6A, B, and Figure 6E, F, respectively. At present, the func- tion of these cells is unknown. Most of the other cells in the hair matrix were not stained distinctly by either antibody. By Nomarski observation, the outer root sheath in the hair bulb was distinguished from the matrix cells (Fig. 6D and H). Figures 7A and D show the staining of hair cuticle (Ce) by anti-myosin antibody. Interestingly this tissue is not stained with anti-actin antibody (Fig. 7B and E). Double- labeling for myosin and actin showed that the hair cuticle was rich in myosin but scarce in actin (Fig. 7C). This obser- vation makes for a marked contrast to the outer root sheath and dermal muscle (Fig. 7C), where both myosin and actin were abundant. The staining for actin in the hair cortex was weak (Fig. 7A). This may suggest possible participation of myosin in the keratinization of the hair shaft without the aid of actin. Nomarski microscopy could distinguish between hair cortex, hair cuticle, inner root sheath (three layers) and outer root sheath (Fig. 7F). IV. Discussion The anagen hair follicle is a minute organ that contains many proliferating and differentiating cells. Hair follicles of rats start to grow from several days before birth, and essen- tially all of them are in the anagen phase at postpartum days 5 to 7. The anagen hair follicle is thus an excellent model for the observation of the morphological processes of cellular differentiation because most cells accumulate in an orderly fashion according to their particular stage of maturation [7, 18]. The cells of the keratinized hair shaft and inner root sheath are apparently elevated passively by the pressure of cell division from underneath. On the other hand, the cells of the hair bulb, outer root sheath, and dermal papillae are thought to move on their own initiative. Our main object was to study the distributions of nonmuscle myosin and its partner, actin, in the hair follicle. For comparative study, skin epidermis and vibrissal follicles were investigated in parallel. The distribution of skeletal muscle myosin was also studied as a reference. Nonmuscle myosin II is known to be widely distributed in species and tissues [3, 9, 11, 13] and seems to function in several types of cells [2–4, 6–8, 22, 24, 25, 28]; however, useful information about myosin and actin in skin/hair is not available. Cross reactions between anti-skeletal muscle myosin antibodies and follicular proteins were reported by Jazwinska et al., with varying results depending on the anti- bodies used [10]. Since their experimental strategy would not be appropriate to obtain definitive results, it is difficult to make a direct comparison between their results and ours. We explain our experimental results as follows: 1) The colocalization of myosin and actin in the spinous layer, as found in the present study, is consistent with the reported results suggesting that protrusion and retraction of lamelli- podia/filopodia are correlated with the dynamism of the myosin and actin molecules [24, 25]. Myosin and actin in the granular layer may be the residues which are slated to disap- pear in the innermost part of the cornified layer. 2) In con- trast to the cells of inner root sheath and hair shaft, those of the outer root sheath undergo cell division at a variety of heights. When the follicle grows downward, the cells of the outer root sheath must move toward the subcutaneous area. The outer root sheath has also been suggested to be the path of stem cells that are known to move upward and downward in the hair follicle [19], while the outer root sheath itself grows in both upward and downward directions depending on its height [12]. Since outer root sheath cells do not under- go terminal keratinization, they are expected to move volun- tarily. It is therefore natural that myosin and actin, which seem to be required for cell migration [1, 22], are found to be localized in the outer root sheath. 3) Since myosin- and actin-rich hair matrix cells are usually adjacent to the dermal papillae, they may play a role in the input and output of nutritional substances and/or hormone-like factors. It is also possible that they are on the verge of moving toward a more distal niche of cell division. Another possibility is that they are melanocytes that have invaded the follicle. Further investigations are needed to verify these views. 4) It is of interest to note that hair cuticle was stained for myosin but not for actin. This suggests that myosin may participate in the distinctive keratinization of the hair cuticle [18]. For example, it may be correlated with the assembly of keratin molecules. 5) The dermal papilla cells are suggested to serve