Arch Dermatol Res (1992) 284:242-245 k a e h ~ o!
9 Springer-Verlag1992
Ultrastructural localization of actin in normal human skin G. Metzler, G. Schaumburg-Lever, B. Fehrenbacher, and H. MiJller Department of Dermatology, Eberhard-Karls-Universitfit, Calwerstrasse 7, W-7400 Tiibingen, Federal Republic of Germany Received October 21, 1991
Summary. Normal human skin was embedded in Lowicryl K4M. Actin microfilaments were localized by applying a postembedding immunogold technique using the monoclonal anti-actin antibody HHF35. Actin microfilaments are part of the cytoskeleton in muscle and nonmuscle cells. Together with myosin they produce contraction. The antibody labelled myofilaments in smooth muscle arrector pill cells, myoepithelial cells and pericytes. In sweat gland cells the microvilli system, a zone beneath the cytoplasma membrane correponding to the adhesion belt region, and apocrine decapitation formations showed labelling. Key words" Immunogold -
Electron microscopy -
H u m a n skin - Actin
Microfilaments are thin filaments about 6 nm in diameter. Together with microtubules and intermediate filaments they make up the cytoskeleton of the cell. As shown by Lazarides and Weber [5] with immunofluorescence techniques actin filaments correspond to the microfilaments observed by electron microscopy. Actin is a 42 k D m o n o m e r called G-actin because of its globular shape. G-actin polymerizes into a fibrous form called F-actin, which corresponds to the microfilaments [1]. Actin is present in both muscle and non-muscle cells [4] and can be subdivided into six isotypes. Skeletal muscle cells express e-actin, smooth muscle cells express e- and 7-actin and non-muscle cells express/~- and 7-actin [9]. In non-muscle cells actin microfilaments have a cytomechanical function. Together with myosin they participate in contractile functions of the cell. The purpose of this investigation was to find out the distribution of actin in normal human skin using the monoclonal antibody H H F 3 5 against actin e- and 7-isotypes [7, 8] and a postembedding immunogold technique. Fig. 1. Myofilaments in myoepithelial cells of sweat glands. M, myoepithelial cell; S, secretory cell. x 12000 Correspondence to: G. Metzler
Fig, 2. Microvillus filament bundle. L, lumen, x 30000
243
Materials and methods
Tissue processing Punch biopsy specimens of normal human skin from the axilla were fixed in 0.l % glutaraldehyde and 2% paraformaldehyde in phosphate-buffered saline (PBS) pH 7.2, dehydrated in ethanol and embedded in Lowicryl K4M (Balzers Union) as described by Carlemalm et al. [2]. Ultrathin sections were mounted on Formvarcoated nickel grids.
Fig. 3. Apical cytoplasma membrane of secretory sweat gland cells. L, lumen, x 20000
Fig. 4. Decapitation formation of apocrine secretion. Dividing membrane (arrows); C, cap and B, base of the formation, • 12000
Fig. 5. Luminal (L) and basal (B) cells of an intradermalduct of sweat glands, x 12000
244
Immunocytochemical procedure For the detection of actin proteins the sections were incubated with PBS for 5 min, PBS containing 0.5% bovine serum albumin for 10min (PBB) and PBS containing 1% goat serum for 10 min, followed by incubation with HHF35 mouse monoclonal antibody against actin (Enzo Diagnostics, New York) diluted 1 : 100 in PBS for 90 rain. The sections were then washed in PBB, incubated with gold-labelled (15 nm) goat anti-mouse IgG (Janssen Biotech, Olen, Belgium) diluted 1:20 in PBS for 60 min, and finally washed in PBB, PBS and distilled water.
Controls for specifity Non-specific binding of anti-actin antibody or the gold-labelled anti-mouse antibody to the embedding resin was evaluated over regions free of tissue. Interaction of the gold-labelled anti-mouse antibody with tissue structures was excluded by omission of the anti-actin antibody.
Results
With the monoclonal antibody HHF35 against actin, actin-binding sites were labelled in eccrine and apocrine sweat gland cells, pericytes and smooth muscle cells. Within sweat glands myoepithelial, secretory and ductal cells showed labelling. Within myoepithelial cells numerous fine filaments running parallel to the long axis of the cell were strongly marked (Fig. 1). In secretory cells a positive reaction product was seen over the entire microvillus filament bundle (Fig. 2). A zone beneath the apical cytoplasma membrane and interdigitating folds of adjacent clear cells were positive (Fig. 3). In decapitation
formations of apocrine secretion, the dividing membrane at the base of the cap reacted with the antibody (Fig. 4). Luminal and basal cells of the intradermal duct showed labelling of filaments lying in the periphery of the cells (Fig. 5). Pericytes showed strong labelling of microfilaments running parallel to the long axis of the cell and reaching into the cytoplasmic processes (Fig. 6). The smooth muscle cells of the arrectores pilorum were filled with bundles of myofilaments that formed dense bodies. Both myofilaments and dense bodies were intensively labelled by the antibody (Fig. 7).
Discussion
It is known that actin is a major component of muscle and non-muscle cells. Together with myosin, actin microfilaments produce contraction. In this study HHF35 labelling was seen over myofilaments of smooth muscle arrector pili cells, pericytes and glandular myoepithelial cells of both eccrine and apocrine sweat glands. The ultrastructural demonstration of actin filaments in myoepithelial cells of human parotid glands has previously been shown by Yoshihara et al. [10]. The strong labelling of filaments running parallel to the long axis of myoepithelial cells suggests a contractile function supporting the secretion of sweat in eccrine and apocrine sweat glands. In capillaries pericytes lie peripherally to endothelial cells [6]. Their long cytoplasmic processes form a discontinuous layer. Myofilaments reach into these processes. Little is known about the function of pericytes. Contract-
Fig. 6. Pericyte, surrounding vascular endothelial cells, x 12000 Fig. 7. Myofilaments of arrector pili smooth muscle cells, x 12000
245 ing myofilaments obviously reduce the cell surface of pericytes. Within sweat gland cells deposits of gold particles were localized at the cell periphery in general, either in small bundles as in microvilli or as a diffuse network in a zone next to the cytoplasmatic aspect of the cell membrane. The labelling of epithelial cells is remarkable since H H F 3 5 antibody is k n o w n to react only with muscle cell actin isotypes [7, 8]. The antibody was assayed by immunocytochemical and biochemical characterization. We found a rather weak but reproducible labelling within sweat gland cells. So the antibody seems to be crossreactive with non-muscle actin isotypes on an ultrastructural level. This assumption is supported by the observation that the reaction product in sweat gland cells is found in those regions of the cells where non-muscle actin is expected to be localized. Epithelial sheets often form a continuous adhesion belt (zonula adherens) around each of the interacting cells of the sheet. The adhesion belt is localized near the apex of each cell just below the tight junction system. As D r e n c k h a l m and Dermietzel [3] were able to demonstrate for the intestinal brush border epithelium, bundles of actin filaments run parallel to the plasma m e m b r a n e and insert into that part of the cell m e m b r a n e belonging to the adhesion belt system. The peripheral labelling of both secretory and ductal glandular cells corresponds with these findings. Thus, this network of actin microfilaments is obviously part of a complex cytoskeletal system stabilizing the shape of the cell.
References 1. Alberts B, Bray D, Lewis J, Raft M, Roberts K, Watson J (1989) Molecular biology of the cell. Garland Publishing, New York/London 2. Carlemalm R, Garavito M, Villinger W (1982) Resin development for electron microscopy and an analysis of embedding in low temperature. J Microsc 126:123-143 3. Drenckhalm D, Dermietzel R (1988) Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study. J Cell Biol 107:1037-1048 4. Hirokava N, Hemser JE (1981) Quick freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells. J Cell Biol 91 : 399-409 5. Lazarides E, Weber K (1974) actin antibody: the specific visualization of actin filaments in non-muscle cells. Proc Natl Acad Sci USA 71:2268-2272 6. Lever WF, Schaumburg-Lever G (1990) Histopathology of the skin, 6th edn. Lippincott, Philadelphia London Mexico City 7. Tsukada T, Tippens D, Gordon D, Ross R, Gown AM (1987) HHF35, a muscle-actin-specific monoclonal antibody 1. Immunocytochemical and biochemical characterization. Am J Pathol 126:51-60 8. Tsukada T, McNutt MA, Ross R, Gown AM (1987) HHF35, a muscle actin-specific monoclonal antibody II. Reactivity in normal, reactive and neoplastic human tissues. Am J Pathol 127:389-402 9. Vandekerckhove J, Weber K (1978) At least six different actins are expressed in a higher mammae. J Mol Biol 126:783-802 10. Yoshihara T, Kanda T, Nagata H, Nomoto M, Kaneko T, Kato Y, Yaku Y (1988) Cytochemical demonstration of actin filaments in myoepithelial cells of the human parotid gland. Acta Anat. 132:317-320
9 Springer-Verlag1992
Ultrastructural localization of actin in normal human skin G. Metzler, G. Schaumburg-Lever, B. Fehrenbacher, and H. MiJller Department of Dermatology, Eberhard-Karls-Universitfit, Calwerstrasse 7, W-7400 Tiibingen, Federal Republic of Germany Received October 21, 1991
Summary. Normal human skin was embedded in Lowicryl K4M. Actin microfilaments were localized by applying a postembedding immunogold technique using the monoclonal anti-actin antibody HHF35. Actin microfilaments are part of the cytoskeleton in muscle and nonmuscle cells. Together with myosin they produce contraction. The antibody labelled myofilaments in smooth muscle arrector pill cells, myoepithelial cells and pericytes. In sweat gland cells the microvilli system, a zone beneath the cytoplasma membrane correponding to the adhesion belt region, and apocrine decapitation formations showed labelling. Key words" Immunogold -
Electron microscopy -
H u m a n skin - Actin
Microfilaments are thin filaments about 6 nm in diameter. Together with microtubules and intermediate filaments they make up the cytoskeleton of the cell. As shown by Lazarides and Weber [5] with immunofluorescence techniques actin filaments correspond to the microfilaments observed by electron microscopy. Actin is a 42 k D m o n o m e r called G-actin because of its globular shape. G-actin polymerizes into a fibrous form called F-actin, which corresponds to the microfilaments [1]. Actin is present in both muscle and non-muscle cells [4] and can be subdivided into six isotypes. Skeletal muscle cells express e-actin, smooth muscle cells express e- and 7-actin and non-muscle cells express/~- and 7-actin [9]. In non-muscle cells actin microfilaments have a cytomechanical function. Together with myosin they participate in contractile functions of the cell. The purpose of this investigation was to find out the distribution of actin in normal human skin using the monoclonal antibody H H F 3 5 against actin e- and 7-isotypes [7, 8] and a postembedding immunogold technique. Fig. 1. Myofilaments in myoepithelial cells of sweat glands. M, myoepithelial cell; S, secretory cell. x 12000 Correspondence to: G. Metzler
Fig, 2. Microvillus filament bundle. L, lumen, x 30000
243
Materials and methods
Tissue processing Punch biopsy specimens of normal human skin from the axilla were fixed in 0.l % glutaraldehyde and 2% paraformaldehyde in phosphate-buffered saline (PBS) pH 7.2, dehydrated in ethanol and embedded in Lowicryl K4M (Balzers Union) as described by Carlemalm et al. [2]. Ultrathin sections were mounted on Formvarcoated nickel grids.
Fig. 3. Apical cytoplasma membrane of secretory sweat gland cells. L, lumen, x 20000
Fig. 4. Decapitation formation of apocrine secretion. Dividing membrane (arrows); C, cap and B, base of the formation, • 12000
Fig. 5. Luminal (L) and basal (B) cells of an intradermalduct of sweat glands, x 12000
244
Immunocytochemical procedure For the detection of actin proteins the sections were incubated with PBS for 5 min, PBS containing 0.5% bovine serum albumin for 10min (PBB) and PBS containing 1% goat serum for 10 min, followed by incubation with HHF35 mouse monoclonal antibody against actin (Enzo Diagnostics, New York) diluted 1 : 100 in PBS for 90 rain. The sections were then washed in PBB, incubated with gold-labelled (15 nm) goat anti-mouse IgG (Janssen Biotech, Olen, Belgium) diluted 1:20 in PBS for 60 min, and finally washed in PBB, PBS and distilled water.
Controls for specifity Non-specific binding of anti-actin antibody or the gold-labelled anti-mouse antibody to the embedding resin was evaluated over regions free of tissue. Interaction of the gold-labelled anti-mouse antibody with tissue structures was excluded by omission of the anti-actin antibody.
Results
With the monoclonal antibody HHF35 against actin, actin-binding sites were labelled in eccrine and apocrine sweat gland cells, pericytes and smooth muscle cells. Within sweat glands myoepithelial, secretory and ductal cells showed labelling. Within myoepithelial cells numerous fine filaments running parallel to the long axis of the cell were strongly marked (Fig. 1). In secretory cells a positive reaction product was seen over the entire microvillus filament bundle (Fig. 2). A zone beneath the apical cytoplasma membrane and interdigitating folds of adjacent clear cells were positive (Fig. 3). In decapitation
formations of apocrine secretion, the dividing membrane at the base of the cap reacted with the antibody (Fig. 4). Luminal and basal cells of the intradermal duct showed labelling of filaments lying in the periphery of the cells (Fig. 5). Pericytes showed strong labelling of microfilaments running parallel to the long axis of the cell and reaching into the cytoplasmic processes (Fig. 6). The smooth muscle cells of the arrectores pilorum were filled with bundles of myofilaments that formed dense bodies. Both myofilaments and dense bodies were intensively labelled by the antibody (Fig. 7).
Discussion
It is known that actin is a major component of muscle and non-muscle cells. Together with myosin, actin microfilaments produce contraction. In this study HHF35 labelling was seen over myofilaments of smooth muscle arrector pili cells, pericytes and glandular myoepithelial cells of both eccrine and apocrine sweat glands. The ultrastructural demonstration of actin filaments in myoepithelial cells of human parotid glands has previously been shown by Yoshihara et al. [10]. The strong labelling of filaments running parallel to the long axis of myoepithelial cells suggests a contractile function supporting the secretion of sweat in eccrine and apocrine sweat glands. In capillaries pericytes lie peripherally to endothelial cells [6]. Their long cytoplasmic processes form a discontinuous layer. Myofilaments reach into these processes. Little is known about the function of pericytes. Contract-
Fig. 6. Pericyte, surrounding vascular endothelial cells, x 12000 Fig. 7. Myofilaments of arrector pili smooth muscle cells, x 12000
245 ing myofilaments obviously reduce the cell surface of pericytes. Within sweat gland cells deposits of gold particles were localized at the cell periphery in general, either in small bundles as in microvilli or as a diffuse network in a zone next to the cytoplasmatic aspect of the cell membrane. The labelling of epithelial cells is remarkable since H H F 3 5 antibody is k n o w n to react only with muscle cell actin isotypes [7, 8]. The antibody was assayed by immunocytochemical and biochemical characterization. We found a rather weak but reproducible labelling within sweat gland cells. So the antibody seems to be crossreactive with non-muscle actin isotypes on an ultrastructural level. This assumption is supported by the observation that the reaction product in sweat gland cells is found in those regions of the cells where non-muscle actin is expected to be localized. Epithelial sheets often form a continuous adhesion belt (zonula adherens) around each of the interacting cells of the sheet. The adhesion belt is localized near the apex of each cell just below the tight junction system. As D r e n c k h a l m and Dermietzel [3] were able to demonstrate for the intestinal brush border epithelium, bundles of actin filaments run parallel to the plasma m e m b r a n e and insert into that part of the cell m e m b r a n e belonging to the adhesion belt system. The peripheral labelling of both secretory and ductal glandular cells corresponds with these findings. Thus, this network of actin microfilaments is obviously part of a complex cytoskeletal system stabilizing the shape of the cell.
References 1. Alberts B, Bray D, Lewis J, Raft M, Roberts K, Watson J (1989) Molecular biology of the cell. Garland Publishing, New York/London 2. Carlemalm R, Garavito M, Villinger W (1982) Resin development for electron microscopy and an analysis of embedding in low temperature. J Microsc 126:123-143 3. Drenckhalm D, Dermietzel R (1988) Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study. J Cell Biol 107:1037-1048 4. Hirokava N, Hemser JE (1981) Quick freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells. J Cell Biol 91 : 399-409 5. Lazarides E, Weber K (1974) actin antibody: the specific visualization of actin filaments in non-muscle cells. Proc Natl Acad Sci USA 71:2268-2272 6. Lever WF, Schaumburg-Lever G (1990) Histopathology of the skin, 6th edn. Lippincott, Philadelphia London Mexico City 7. Tsukada T, Tippens D, Gordon D, Ross R, Gown AM (1987) HHF35, a muscle-actin-specific monoclonal antibody 1. Immunocytochemical and biochemical characterization. Am J Pathol 126:51-60 8. Tsukada T, McNutt MA, Ross R, Gown AM (1987) HHF35, a muscle actin-specific monoclonal antibody II. Reactivity in normal, reactive and neoplastic human tissues. Am J Pathol 127:389-402 9. Vandekerckhove J, Weber K (1978) At least six different actins are expressed in a higher mammae. J Mol Biol 126:783-802 10. Yoshihara T, Kanda T, Nagata H, Nomoto M, Kaneko T, Kato Y, Yaku Y (1988) Cytochemical demonstration of actin filaments in myoepithelial cells of the human parotid gland. Acta Anat. 132:317-320
