Embryonic day 18 (E18) rat epidermis contains antigens for a number of antibodies previously thought to be specific for particular sarcomeric MyHC isoforms (Fig. , see also Table for a summary of antibodies, their known sarcomeric MyHC epitopes and their reactivity with non-muscle tissue). Among a set of eight monoclonal antibodies raised against purifed human skeletal muscle myosin [6
] we found that A4.840, N3.36 and N2.261 detected subsets of cells in E18 epidermis. The A4.840-antigen could be detected in all layers of epidermal cells in cryosections of E18 rat skin (Fig. ). In contrast, N3.36-antigen was present only within a subset of cells in the basal layer (Fig. ). A third anti-skeletal muscle MyHC antibody, N2.261 also weakly detected cells in basal epidermis (Fig. ). At this stage of development, A4.74- and A4.951-antigens were barely detectable (Fig. ). Control IgM- and IgG-class anti-sarcomeric MyHC antibodies A4.1519 and F1.652 showed no reaction to epidermis, indicating that binding of A4.840, N3.36 and N2.261 was specific (Fig. , respectively). Skin antigens are not identical to skeletal muscle MyHCs because monoclonal antibodies A4.1025 and MF20 did not react with epidermis (Fig. and data not shown), even though each is reported to detect all isoforms of sarcomeric MyHC [12
]. Ten further mouse monoclonal antibodies against sarcomeric MyHC isoforms did not react with rat epidermis at any age examined. In particular, BA-D5, BA-F8 and NA8 antibodies, all of which detect slow skeletal MyHCs in a variety of vertebrate species [6
], failed to show any reactivity with epidermis (data not shown). Considering that A4.840- and N3.36-epitopes are normally expressed on the products of different sarcomeric MyHC genes, the data above show that epidermis contains molecules antigenically-related to, but distinct from, sarcomeric MyHC. Hereafter we refer to these molecules as 'MyHC-like' simply to denote this antigenic similarity.
Figure 1 Distinct MyHC-like proteins are expressed in different subsets of embryonic rat epidermal cells. Transverse cryosections of E18 rat lower hindlimbs were allowed to react with monoclonal antibodies raised against human skeletal muscle myosin and detected (more ...)
Summary of antibodies, their known MyHC epitopes and epithelial reactivity.
To determine when and in which cells the MyHC-like molecules are expressed, we analysed cryosections throughout postnatal epidermal development when the skin thickens and matures and hair follicles are formed, and found additional sarcomeric MyHC antibody cross-reactivities. By postnatal day 1 (P1) A4.840-antigen was present in the full thickness of the suprabasal surface epidermis and also within many cell layers of the hair follicle (Fig. , shown diagrammatically in ). N3.36-antigen was restricted to a subset of basal cells in both surface epidermis and an outer layer of the hair follicle (Fig. ). In contrast to younger skin, in which hair follicles had not yet begun to mature, we observed an A4.74-antigen in a single layer of cells near the base of the growing hair follicles (Fig. ). By P1, N2.261-antigen detected all basal layer cells in surface epidermis, both those that express N3.36-antigen and those that do not (Fig. ). However, N2.261-antigen was not detected in the hair follicle (Fig. ). The N1.551-antigen was found restricted to a small cluster of cells in the neck of hair follicles (Fig. ). Many other anti-MyHC antibodies, such as A4.1025, F1.652 and A4.1519, do not show any reactivity with skin (data not shown). So, the patterns of expression of the MyHC-like molecules change with development in ways that suggest each may have specific functions in particular epidermal cell types.
Figure 2 MyHC-like molecules are developmentally regulated in skin. Cryosections of P1 rat back skin was reacted with A4.840 (A), N3.36 (B), A4.74 (C), N2.261 (D) or N1.551 (E) and the antibody detected by peroxidase immunohistochemistry. Hair follicles were sectioned (more ...)
We examined the epidermis of species other than rat, on the rationale that evolutionarily conserved epitopes are likely to have functional significance. A4.840, N3.36 and N2.261 showed similar staining in P5 rat back and adult human scalp epidermis (Fig. ). A4.840 detected most cells in epidermis, although more weakly as the skin cornified, and in the hair follicle (Fig. ). In contrast, N3.36 detected a few cells of the basal layer of P5 rat epidermis, but not adult human surface epidermis. However, the outer layers of the hair follicle were labelled in both species (Fig. ). As in rat, in human tissue A4.74 only reacted with cells in the inner root sheath of hair follicles, and N2.261 reacted with basal cells (compare Fig. and ). In mouse skin, A4.840 and A4.74 reacted with similar, if not identical, sets of cells to those detected in the rat (Fig. ). However, N3.36 did not detect proteins in mouse skin (Fig. ). This again suggests that the molecules in skin are not the same as those in muscles, because N3.36 reacts to mouse sarcomeric MyHC, just as to rat and human. We have also observed A4.840-reactivity in chicken skin and feather germs (data not shown). Taken together, these data suggest a family of MyHC-related molecules with substantial evolutionary conservation are expressed in epidermis.
Figure 3 The MyHC-like molecules are evolutionarily conserved and found in a variety of epithelial tissues. Cryosections of P5 rat tail skin (A-C), adult human scalp skin (D-G), P3 mouse back skin (H-J) and adult rat jejunum (K-M) were reacted with A4.840 (A,D,H,K), (more ...)
The MyHC-like proteins are also expressed in other epithelial tissues
To shed light on the potential functions of the MyHC-like molecules in epidermis, we analysed a variety of epithelial and non-epithelial tissues for the presence of the MyHC-like molecules. We never observed any reaction with any non-epithelial tissues other than skeletal or cardiac muscle, consistent with the fact that the monoclonal antibodies to specific skeletal muscle isoforms were originally screened for lack of reaction with several non-muscle tissues [19
]. However, we did find reactivity with several epithelial tissues. A4.840 reacted broadly with all cells of the gut epithelium, whereas N3.36 reacted in a small cluster of cells at the base of each crypt (Fig. ). A4.74-antigen was undetectable in gut (Fig. ). N3.36 reacted with the simple epithelium of the bladder (data not shown). N3.36, N2.261 and A4.840 each reacted with distinct cell populations in the non-neural retina (data not shown). None of these tissues reacted with the general anti-sarcomeric MyHC antibody A4.1025 (data not shown). Thus, the new antigens may perform similar functions in several epithelial cell populations.
Sub-cellular localisation of the MyHC-like molecules
Myosin IIs are generally cytoplasmic proteins that can form filaments and generate force in concert with actin. To determine whether the antigens we detected might have a similar role, we examined the sub-cellular location of each antigen. Initial analyses of cryosections using immunohistochemistry and fluorescence showed that A4.840-reactive molecules were at the cell periphery, whereas A4.74- and N3.36-reactive molecules seemed to accumulate more widely within the cytoplasm (Figs. ,). To obtain better spatial resolution of molecules within epidermal cells, we developed a method of partial trypsinisation which loosened the connections between cells while retaining their relative positions within the tissue. Cryosections of this trypsinised material gave good spatial resolution and clearly revealed that A4.840-reactivity was located at the plasma membrane in a punctate distribution (Figs. ). The punctate distribution of A4.840-reactive material close to the plasma membrane was highly reminiscent of the abundant desmosomes of epidermis [20
]. We employed anti-desmoplakin antibodies in dual immunofluorescence confocal microscopy to examine the possibility of co-localization more closely (Fig. ). Using fresh frozen cryosections which had not been subjected to trypsinisation, we confirmed the punctate distribution of A4.840-antigen and found it to be strongly associated with desmoplakin immunoreactivity at cell borders (Fig. ). We compared images of surface epidermis A4.840-labelling with those of desmoplakin-labelling (Fig. ) and scored the number of puncta of fluorescence that labelled with one, the other or both antibodies. Greater than 90% of puncta showed co-localization of A4.840-antigen and desmoplakin. We obtained two pieces of evidence in favour of an intracellular location for the A4.840-antigen: a) A4.840 showed no punctate staining in wholemount stains of unpermeabilised P7 rat skin but readily labelled the surface of hair follicle cells in punctate distribution in wholemount skin fragments permeabilised with Triton X100 (Fig. ), b) punctate A4.840-reactivity could be detected in permeabilized cultured skin cells (see Fig. ), but were not detected when cells were stained without fixation or permeabilization (data not shown). Attempts to immunolocalise the A4.840-antigen by electron microscopy have so far been unsuccessful, probably due to fixation sensitivity of the A4.840 epitope (unpublished observations kindly provided by Alison North and David Garrod). We conclude that A4.840-antigen is co-localized with desmosomes.
Figure 4 Cellular and sub-cellular localisation of A4.840-antigen. Immunofluorescent detection of A4.840 (A,C,E,F red; G,I green), A4.74 (B,D, green), and desmoplakin (E,F, green; H,J, red) in rat epidermis. A-D. A4.840-epitope is distributed at the cell periphery (more ...)
Figure 5 Distribution of MyHC-like antigens is retained in epidermal cells in tissue culture and mimicked in cardiomyocytes. Primary dissociates of P1 rat skin (A-D) were grown in culture and stained for A4.840 (A) or N3.36 (C) and A4.74 (B,D) by dual immunofluorescence. (more ...)
Desmosomes are not only present in epithelia but also in other tissues under tensile stress. For example, in the myocardium, desmosomes are found at specialised cell-cell contacts, the intercalated disks, where cardiomyocytes are tightly connected to ensure mechanical coupling during the contraction process. Primary cultures of cardiomyocytes continue to beat and intercalated disk-like structures are re-established. The A4.840 antigen co-localized with desmoplakin, plakoglobin and pan-cadherin antigens at these intercalated disk-like structures (Figure ). Additional A4.840 reactivity was found in the myofibrils (Figure ), localized in the region of the M-band of the sarcomere, as demonstrated by double-labelling with antibodies to myomesin, an integral component of the M-band that is responsible for integrating myosin and titin filaments in the sarcomere [22
]. However, only some cardiomyocytes showed these sarcomeric striations with the A4.840 antibody and there are myomesin-stained M-bands that do not show the A4.840 signal (asterisk in H-J). Double staining of the A4.840 antigen with the muscle intermediate filament protein desmin, which is localized around the Z-disks of the sarcomere, revealed alternating striations of the myofibrils (Figure ). Taken together, these data raise the possibility that in cardiac muscle, in addition to A4.840-reactive slow sarcomeric MyHC, the novel A4.840 antigen is also expressed and associated mainly with the intercalated disk.
Unlike the A4.840-antigen, A4.74-reactivity was distributed in cells of the hair follicle in a fibrillar arrangement (Fig. ). Dual stained sections showed that A4.74-epitope was present in the outermost layer of the A4.840-epitope-containing cells of the hair follicle just above the bulb (Fig. ). Similar comparisons between N3.36- and A4.74-antigens showed each to be present within a distinct concentric layer of cells in the follicle, with N3.36 reacting with cells further from the hair itself (Fig. ). Both antigens were cytoplasmic (Fig. and Fig. ). No extracellular or nuclear labelling was detected with any of the three antibodies. Thus, the MyHC-like molecules are distributed distinctly within cells.
Figure 6 Association of the MyHC-like molecules with intermediate filament-containing structures. N3.36-antigen (A,B,F red; D green), A4.74-antigen (A-C, green; G, red), A4.840-antigen (C, red), phalloidin (E), pan-keratin (F, green) and keratin 5 (G, green) in (more ...)
The A4.74- and N3.36-antigens were localized in fibrillar structures within the cytoplasm of fibres (Fig. and Fig. ). To characterize their distribution in more detail we tested whether these fibrillar structures represented one of the described cytoskeletal fibrillar systems. Neither N3.36- nor A4.74-reactive material was co-localized with actin filaments labelled with rhodaminated-phalloidin (Fig. and data not shown). On the other hand, A4.74-antigen localized to filaments within the cytoplasm of the hair follicle cells that appeared to connect desmosomes, revealed by abundant A4.840-antigen (Fig. ). As desmosomes are connected in this manner by keratin filaments in epidermis [23
], this finding suggested that A4.74-antigen may be keratin intermediate filament-associated. The sub-cellular distribution of N3.36-antigen was very similar to that revealed by an anti-pan-keratin antibody in the outermost layer of the root sheath (Fig. ), although not all keratin-reactive material co-localized with N3.36-antigen within these cells. Based on dual staining for A4.74-antigen and keratin 5, it appears that N3.36-antigen is expressed in a subset of cells that express keratin 5 (Fig. ). Taken together, these data suggest that both the A4.74-antigen and N3.36-antigen are co-localized with intermediate filament proteins in the hair follicle.
To analyse antigen location in more detail, we examined primary dissociated cells from P1 rat skin that had been grown in culture for two days. A4.840-antigen was often, but not exclusively, localized in plasma membrane-associated dots in the cultured cells (Fig. ). Both the A4.74- and N3.36-antigens remained in fibrillar structures within distinct populations of these dissociated and fixed cultured epidermal cells (Fig. ). Whereas A4.74-antigen was in filaments in either large oval or small round cells in these primary dissociates, most A4.840-antigen that we could detect was located in large cells with a morphology reminiscent of cells in the prickle cell layer of our partially trypisinised tissue (Fig. ). In contrast, N3.36-antigen was in a distinct population of cultured cells not labelled with A4.74 (Fig. ), just as we observe in vivo. The numbers of cells expressing the MyHC-like antigens appeared to decline with time in dissociated cell culture, suggesting the loss of tissue integrity or cell-cell contact may lead to loss of the antigens. However, due to the small numbers of antigenically-reactive cells, we could not eliminate the possibility of cell death accounting for this observation (data not shown). Consistent with the idea of loss of MyHC-like antigens in culture, after two days in vitro we observed A4.74-reactive cells that did not contain A4.840-antigen (Fig. ). Such cells were never observed in vivo. Examination of differentiating human epidermal keratinocyte cultures also failed to reveal any of the MyHC-like antigens, even though differentiation-marker keratins were expressed and desmosomes were formed (data not shown). These results suggest that expression or accumulation of the MyHC-like antigens, requires some aspect of the in vivo environment that is not mimicked in the culture systems examined.
Molecular characterisation of A4.74-antigen
To gain further insight into the nature of the MyHC-like antigens, we performed a partial purification. Western analysis of P9 rat tail skin homogenates revealed a protein of around Mr 230000 that could be detected by antibody A4.74 (Fig. ). Antibodies A4.840 and N3.36 did not detect any protein in skin homogenates, despite detecting slow and fast MyHCs (respectively) from muscle tissue, and so could not be analysed further (data not shown). However, the lack of reactivity of these antibodies on skin Western blots supports the notion that the skin and muscle antigens are distinct. The detected skin A4.74-antigen was not a conventional skeletal muscle MyHC because it migrated more slowly than muscle MyHCs and it did not react with antibodies, such as A4.1025, that detect all isoforms of skeletal muscle MyHC (Fig. ). Although no obvious band was visible on Coomassie Blue-stained gels at the position of the A4.74-antigen, the antibody reacted strongly with this Mr 230 000 band, even in comparison to its reactivity with muscle MyHC (Fig. ). Moreover, the skin sample did not contain skeletal muscle, as antibody A4.1025 does not detect any protein in tail skin, although it readily detects MyHC in skeletal muscle (Fig. ). A4.1025 does detect MyHC in proteins isolated from P7 rat back or head skin because these tissues are usually contaminated during isolation with the underlying thin dermal muscle layer, the paniculus carnosus, which is not present in the tail (data not shown and Fig. ). Other antibodies that detect either all skeletal MyHCs or subsets of these proteins do not react with the skin sample. For example, F1.652, an antibody which recognizes embryonic mammalian MyHC, does not detect proteins in rat skin (Fig. ). Thus, skin contains a skeletal muscle fast IIA MyHC-related protein that is not identical to the muscle myosin.
Figure 7 Skin A4.74-antigen is distinct from fast skeletal myosin IIA and co-purifies with keratin. A. Western analysis of P9 rat tail skin proteins (Sk) shows that proteins around Mr 230000 are detected by A4.74, but not by F1.652 or A4.1025. A4.74 detects a (more ...)
We obtained further evidence that the A4.74-antigen in skin was not skeletal muscle fast IIA MyHC. When skeletal muscle is homogenised in high salt solutions myosin is readily extracted. Similar homogenisation of skin failed to release the A4.74-antigen (data not shown). Indeed, homogenisation by mechanical, manual or sonication procedures in solutions containing high salt with or without various detergents failed to release the A4.74-antigen from material that could be pelleted by centrifugation at 100,000 × g for 15 minutes. In contrast, solubilisation of tissue in buffer containing 6 M urea did release the A4.74-antigen to the high speed supernatant (Fig. ). Subsequent dialysis against PBS caused a re-precipitation of various highly insoluble skin proteins, including keratins, and this procedure also quantitatively precipitated the A4.74-antigen (Fig. ). Dialysis into 0.6 M KCl-containing buffer, which would retain sarcomeric myosin in the supernatant, did not affect the precipitation of the A4.74-reactive protein in skin (data not shown). Therefore, the A4.74-reactive 230 kDa protein in skin co-purifies with skin keratins, and is not skeletal fast IIA MyHC.