J Mel Cell Cardiol
23. 873-882 (1991)
Persistence of an Embryonic Intermediate Filament-associated in the Smooth Muscle Cells of Elastic Arteries and in Purkinje Michel
Vincent*,
CHUL
Sylvain
Levasseur,
R. William
Currief-
and Peter
Protein Fibres A. Rogers
Research Center and Department of Medicine, Lava1 University, Ste-Foy, Quk%ec, Canada (Received 14 August 1990, accepted in revisedform 7March 1991)
M. VINCENT, S. LEVASSEUR, R. W. CURRIE AND P. A. ROGERS. Persistence of an Embryonic Intermediate Filament-associated Protein in the Smooth Muscle Cells of Elastic Arteries and in Purkinje Fibres. Journal of Molecular and Cellular Cardiology (1991) 23, 873-882. During differentiation of most myogenic tissues, vimentin is transiently expressed as the intermediate flament (IF) protein subunit and is progressively replaced by desmin. However, smooth muscle cells of mature vascular tissue contain variable amounts of vimentin whose cellular content decreases as function of the distance from the heart. IFAPa-400 is a developmentally regulated IF crosslinker protein whose expression appears to parallel that of vimentin during chick myogenesis. Immunohistological and immunoblot techniques were employed to study the expression of this protein in cardiac and vascular smooth muscle cells of the adult chicken. As observed for vimentin, the expression of IFAPa-400 persists in the mature smooth muscle cells of the large arteries as they leave the heart. However, both of these proteins are downregulated according to a proximo-distal gradient with respect to the distance from the heart. Conversely, desmin is much more abundant in the distal segments of the aorta. Thus, the co-ordinate expression of vimentin with IFAPa-400 may be a characteristic feature of elastic vascular tissue in which it could meet mechanical requirements close to those of the embryonic cells expressing them. This hypothesis is supported by the observation that the single cell type which continues to express the vimentin-IFAPa-400 combination in the mature heart is the Purkinje fibres, which are also subjected to high mechanical tensions but in which myofibrils are generally sparse compared to working myocytes KEY WORDS: Vascular smooth filaments; Vimentin; IFAPa-400;
muscle cells; Elastic Desmin; Chicken.
Introduction Intermediate filaments (IF) constitute a class of cytoskeletal filaments which are morphologically and biochemically distinct from microfilaments and microtubules. The protein subunits of IF vary according to the cell type and this specificity led to the following system of classification: cytokeratins in epithelial cells, vimentin in mesenchymal cells, desmin in muscle cells, glial fibrillary acidic protein (GFAP) in glial cells and neurofilaments in neurones [12, 261. As the primary sequences for many IF proteins became available, a more precise classification into five distinct sequence types was proposed based on size and sequence differences in the central o-helical-rich rod domain whose conserved secondary structure accounts for the high degree of similarity in IF morphology [20]. *Author Boulevard I Present
to whom Laurier, address:
0022-2828/91/070873
arteries;
Atherosclerosis;
fibres;
Intermediate
Vimentin, desmin and GFAP share the IF type III sequence features. Desmin and GFAP are expressed in mature muscle cells and astrocytes respectively whereas vimentin, typically found in cells of mesenchymal origin, has also been reported in various cultured cells [26]. In addition, vimentin is known to be transiently expressed during muscle and nerve cell differentiation where it is gradually replaced by a second IF protein, characteristic of the mature cell type [9, 24, 26, I]. Vimentin is therefore unique in this respect since its expression appears to be regulated according to either celI or developmental type programme. We have recently identified and partially characterized a high molecular weight (MW) IF-associated protein, IFAPa-400 [21], whose expression is confined to the myogenic and the
correspondence should be addressed: Ontog6nese Ste-Foy, QuCbec, Canada GlV 4G2. Department of Anatomy, Dalhousie University, + 10$03.00/O
Purkinje
et GtnCtique Halifax,
Moltculaire Nova
Scotia,
CHUL,
2705
Canada.
0 1991 Academic
Press
Limited
a74
M. Vincent
neurogenic tissues of the chick embryo, i.e. the cell lineages that transiently express vimentin in development [5, s]. Due to this transient coexpression, we believe that IFAPa-400 is involved in the structural re-organization of the IF during muscle and neural cell differentiation. The vascular smooth muscle cells differ from the other myogenic lineages in that their IF may contain various amounts of vimentin in addition to desmin [8, 7,191. In mammals the ratio of vimentin to desmin may be related to the elasticity of large arteries. This ratio is higher in large arteries such as the aorta than in muscular arteries [ 7, 131. Since vimentin is expressed in some vascular smooth muscle cells and IFAPa-400 is transiently expressed during the developmental shift of vimentin to desmin in the other myogenic lineages, we decided to investigate whether this giant protein persists with vimentin in the elastic arteries. Here we report the persistence of IFAPa-400 in the vascular smooth muscle cells of the adult chicken; its expression is parallel to that of vimentin and the reversed of that of desmin. We also describe the persistence of IFAPa-400 in a cellular subpopulation of the mature heart, the IF-enriched Purkinje fibres.
Materials and Methods Six-month-old White Leghorn hens and fertilized eggs were purchased from a local hatchery. The eggs were maintained in a humidified forced-air incubator (80 to 90% relative humidity) at 38% and embryos were classified according to the number of incubation days.
Preparation
of antibodies
The monoclonal antibody (mab) F51H2 (IgM) was obtained by immunization of a Balb/c mouse with early differentiating chick embryo somites as described previously [271. The infilament-associated protein termediate (IFAPa-400) identified by this monoclonal was purified by ion exchange chromatography of embryonic heart ionic an high strength/detergent-resistant fraction solubilized in 8~ urea, 1OmM Tris-HCl, pH 7.4. The fractions containing IFAPa-400 reactivity were pooled, dialysed against 1OmM Tris-HCl, pH 7.4 and resolved by SDS-PAGE. A poly-
et al clonal antibody to IFAPa-400 was raised by injecting a rabbit with the IFAPa-400 band excised from acrylamide gels. The antibody was rendered monospecific by triple absorption of the serum on the protein immobilized on nitrocellulose. In order to validate our results, we used several monoclonals obtained after immunizing mice with IFAPa-400 prepared by ion exchange chromatography. Those from the IgG subclass showing immunofluorescence and immunoblot spatiotemporal patterns of reactivity identical to F51H2 were selected. A monoclonal antibody against vimentin (6G2) was generated using chicken erythrocyte vimentin as an antigen [5]. A rabbit polyclonal antiserum was raised against electrophoretically purified chicken desmin and has been previously characterized
WIGel electrophoresis and immunoblotting Hearts from embryonic and adult chickens were excised immediately following sacrifice, rinsed in PBS and frozen in liquid nitrogen. The aorta was dissected from the base of the heart to the abdominal bifurcation and further divided into proximal (ascending), intermediate (thoracic) and distal (abdominal) portions, then rinsed in PBS and frozen in liquid nitrogen. Frozen tissue samples were stored at - 80°C until electrophoretic analysis. Samples were homogenized in SDS sample buffer (2.3% SDS, 5% fimercaptoethanol, 10% glycerol, 0.062~ Tris pH 6.8) containing a cocktail of protease inhibitors (1mM phenylmethylsulphonyl fluoride, 5gglml leupeptin, 5pglml pepstatin, sag/ml antipain) and heated at 95°C for 3 min. Samples were then resolved by SDSPAGE on 3-12s or 7-15s gradient gels with an acrylamide: bis ratio of 30:0.15 in Laemmli’s buffer system [11]. After electrophoresis, the proteins were transferred electrophoretically to nitrocellulose at 200 mA overnight [25] and the MW markers visualized by staining with rouge Ponceau [4]. The nitrocellulose sheets were saturated with PBS containing 5% powdered milk [IO] before incubation with a primary antibody for lh at 37’C. Both ascitic fluid (mouse monoclonals F51H2 and 6G2) and rabbit antiserum were used at a dilution of 1: 1000. After extensive washes in PBS containing 0.05% Tween 20, the blots
875 were treated with either *251-labelled goat IgGs antimouse or antirabbit immunoglobulins. After washing, the blots wefe dried and exposed to Kodak X-Omat AR film at 80‘33 with intensifying screens.
Histologicalprocedures Ether-anaesthetized hens were perfused by cannulation of the left ventricle using heparinized PBS, 10% formalin and Perfix (Fisher Scientific) in sequence. The heart and the aorta were then dissected into small pieces and fixed overnight in Per-fix. After extensive washing in PBS, the pieces were progressively dehydrated in ethanol and toluene and embedded in a mixture of beeswax-Paraplast (1:8). Sections (5-7pm thick) were cut and mounted onto gelatin-coated glass slides. Before immunolabelling, the sections were deparaffinized in toluene, rehydrated through a decreasing ethanol series and washed several times in PBS.
Indirect immunoJuorescence Immunostaining on tissue sections was performed using monoclonal antibodies either as pure hybridoma supernatants or 1:50 dilution of ascitic fluids in PBS containing 0.5 % BSA and rabbit polyclonal antibodies at a dilution of 1:50. Sections were incubated in the primary antibody for 45 min at room temperature. After several PBS rinses, the sections were incubated for 45 min with FITCconjugated goat IgG antimouse immunoglobulins to detect monoclonal antibodies or with TRITC-conjugated goat IgG antirabbit immunoglobulins to reveal the polyclonal antibodies. In double-labelling experiments, primary antibodies (a monoclonal and a rabbit polyclonal) were mixed together before incubation and revealed as above. Mismatched incubations revealed no cross-reactivities between the two detection systems. Negative controls were routinely made by incubating the tissue sections either with irrelevant monoclonals or PBS containing 0.5% BSA. The specimens were mounted with p-phenylenediamine-PBS in glycerin [II] and observed under a Zeiss Axiophot microscope equipped with FITC and TRITC filter sets.
Results
Expressionof IFAPa-400 in adult aorticsmooth musclecells As part of our study of the developmental modulation of IFAPa-400 in chicken myogenic tissues, immunoblot experiments were carried out on whole homogenates from cardiac and aortic tissues explanted at different developmental states (Fig. 1). The expression of IFAPa-40 dramatically decreased during cardiogenesis as observed with skeletal and visceral smooth muscle. With equivalent amounts of proteins loaded on the gels, the 400 kD immunoreactive band was maximal at embryonic day (ED) 12, less intense at hatching (ED 21) and barely detectable in the adult. This pattern of immunoreactivity was completely reversed in the ascending aorta (Fig. 1). In this case, IFAPa-400 was hardly detectable in the first 10 or 11 days of incubation but was abundant in older embryos and in adult chickens. The same results (not shown) were obtained using the polyclonal antibody raised against IFAPa-400 and therefore 7
12
16
21
A
7
12
16
21
A
-240Kd - 220
- 116 -
94
-68
-
Heort
42
Aorto
FIGURE 1. Expression of IFAPa-400 during heart and aorta embryogenesis. Immunoblot analysis of total tissue extracts from the heart and the ascending aorta prepared at different developmental stages and reacted with mab F51H2. Approximately equal amounts of proteins were loaded in each well. The expression of IFAPa-400 drastically decreased in the heart during development but increased in the aorta. The numbers represent the embryonic days. A, adult. The MW markers are shown in the margin.
876
FIGURE 2. Co-localization sections were double-labelled 6G2 respectively. IFAPa-400 the vimentin-rich connective (185 x )
M. Vincent
et al
of IFAPa-400 and vimentin in the smooth muscle cells ofthe ascending aorta. Transverse for IFAPa-400 (a) and vimentin (b) using the affinity-purified polyclonal antibody and mab co-localized with vimentin in the smooth muscle cells (bright layers) but was absent from tissue cells [in~l&~&~r punctatc staining in (b)] and from the endothelial cells (arrows).
demonstrated that IFAPa-400 down-regulation observed during myogenesis and neurogenesis does not occur during aorta ontogenesis. Since IFAPa-400 expression appeared closely related to that of vimentin during ontogenesis, we investigated whether its distribution in a mature tissue coincided with that of vimentin. Using double immunofluorescence, IFAPa-400 was shown to co-localize with vimentin in the smooth muscle cells of the ascending aorta but was absent from the vimentin-positive fibroblast-like cells dispersed in the interlamellar connective tissue layers (Fig. 2). In this large vessel, vimentin was also shown to be enriched in endothelial cells but no IFAPa-400 could be detected in this cell type (arrows in Fig. 2). It appears therefore that IFAPa-400 is not an obligatory companion of vimentin and that its expression in the adult aorta is confined to the smooth muscle cells.
Topopafihic
modulation of IFAPa-400 expression in the aorta
and IF
Three contiguous segments of the aorta and the pulmonary artery were examined by immunoblot to determine whether gradients of expression of IFS occurred along the length of the avian large arteries (Fig. 3) The segment of the aorta most proximal to the heart was excised just above the aortic valve and included the aortic arch. The intermediate portion corresponded to the aorta thoratalis whereas the most distal part comprised the beginning of the aorta abdominalis down to the renal arteries. The proximal portion of the pulmonary artery consisted of the common trunk originating from the right ventricle, the intermediate portion included the right and left pulmonary division and the fraction corresponding to the most distal segment included the intrapulmonary ramifications which we carefully dissected free from adjacent tissues.
877
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- nebulln
-24OKd - 220
- 116 -64 -66 - 42
Ylrul - 42 -66
DES
,-q
-Q
c
- 42
FIGURE 3. Variations of IFAPa-400 and IF expression along the aorta and the pulmonary artery. Immunoblot analysis of total tissue extracts prepared from three contiguous segments of the aorta and the pulmonary artery and reacted with mabs F5lH2 and 6G2 and with the antidesmin serum. In each case, the extracts from the heart most proximal segments arc on the left lane (pmx). Compared to the typical IF composition of the mature muscle cell in which desmin (ties) was abundant and vimentin (vim) and IFAPa-400 were faint (heart, first lane), the segments of both arteries most proximal to the heart contained high concentrations of IFAPa-400 and vimentin and little desmin. The most distal segments were enriched with desmin and contained little or no vimentin and IFAPa-400. The MW markets ate shown in the margin. Rat skeletal muscle extracts were run in the top gel to provide additional high MW markers for IFAPa-400 MW determination. (titin z 226OkD; nebulin 6008OOkD)
vimentin-rich most proximal parts (Fig. 3). Thus the IF composition of the muscular wall of the large arteries coming from the heart undergoes rapid changes along their length. Those portions closer to the heart contain predominantly vimentin which presumably is associatedwith IFAPa-400 and trace amounts of desmin whereasthe most distal segmentsare more representative of the other mature muscle tissuesin that they are enriched with desmin but contain little or no vimentin and IFAPa-400. This typical IF composition of the mature muscle can be visualized in the heart (Fig. 3, first lane) in which desmin is abundant while IFAPa-400 and vimentin are barely detectable (seebelow). To verify whether the quantitative changes in the aorta IF composition arose from variations in the number of cells expressing these proteins, immunofluorescence microscopy was performed on aortic transverse sections at different levels. As shown above (see Fig. 2), IFAPa-400 and vimentin co-localized in the smooth muscle cellsof the proximal part of the aorta. At the level of the iliac bifurcation, however, double-labellings using the afhnitypurified rabbit antiserum to IFAPa-400 and the monoclonal 6G2 antivimentin revealed that both proteins were much lessabundant in the smooth muscle cells (Fig. 4). Also consistent with the immunoblot pattern, the desmin fluorescence was strong [Fig. 5(b)]. Although vimentin reactivity was weak in the smooth muscle cells, sporadic staining was observed either in the endothelial cells [arrow in Fig. 4(a)] or in putative mesenchymal components [Fig. 5(a)]. IFAPa-400
and vimmtin
in heart PurkinjeJibres
Immunoblot analysis of adult cardiac tissue using various IFAPa-400 antibodies consistently showed a faint immunoreactive band at the apparent molecular weight corresponding to IFAPa-400 ( see first lane in Fig. 3). Extreme care was taken to avoid any contamination by the outcoming arteries. Immunofluorescence staining of adult heart sections demonstrated the absence of In both arteries, parallel immunoblot ex- IFAPa-400 immunoreactivity in ordinary periments using F51H2, monoclonal6G2 anti- myocardial cells but a very high level in a vimentin and the antidesmin serum revealed subset of cells primarily located around blood opposite gradients for vimentin and desmin vessels (Fig. 6) and underneath the and high concentrations of IFAPa-400 in their endocardium (arrows in Fig. 7). These large
FIGURE 4. Absence of IFAPa-400 aorta at the level of the lilac bifurcation proteins was very faint in the smooth (arrow). (340 x )
and vimentin in the smooth muscle cells of distal aorta. Transverse sections of ‘the were double-labelled for vimentin (a) and IFAPa-400 (b). The staining for 1:10th muscle cells but some vimentin reactivity could be seen in the endothelial s:ells
FIGURE 5. Expression of desmin in the smooth muscle cells of the distal aorta. Transvene sections of the aorta at the level of the iliac bifurcation were double-labelled for vimentin (a) and desmin (b). The smooth muscle cells exhibit st rong desmin fluorescence. Vimentin reactivity is confined to some putative mesenchymal components. (350 x )
IFApa-400
in Vascular
Smooth
Muscle
heart FI( 3URE 6. Expression of IFAPa-400 in the Purkinje fibres of the mature heart. Sections of the adult chicken labelled for IFAPa-400 with the monoclonal antibody F51H2. Strong fluorescence was observed in pu tative cells. clustered around a blood vessel. Note the absence of reactivity in the surrounding myocardial Purki ln,je fibres (500 X)
FI’ were unde cells, (490
GURE labelled ,meath which X)
7. Expression for IFAPa-400 the endocardium may represent
I heart of IFAPa-400 in the Purkinje fibres of the mature heart. Sections of the adult thicker with the monoclonal antibody F51H2. Purkinje fibres forming one-cell thick s trands were intensely labelled (arrows). Some fluorescence was also observed in the surrou mding leads). a transitional form between Purkinje fibres and working cardiac myocytes (arrowh
880
M. Vincent
cells have centrally located nuclei and occasionally a few myofibrils can be observed arranged peripherally around central refringent cytoplasmic material. These cells have been previously identified in the chicken heart as Purkinje fibres [18]. When labelled with desmin antibody, these Purkinje fibres were more fluorescent than the surrounding myocytes (data not shown). Double-labelling heart sections with polyclonal anti-IFAPa-400 and monoclonal antivimentin demonstrated the coincidence of both proteins in the Purkinje fibre cytoplasm. In contrast, other vimentin-positive cells including endothelial cells and fibroblasts were devoid of IFAPa-400 (data not shown). Although IFAPa-400 could not be detected in ordinary myocytes, some staining was occasionally observed in subsets of cells usually close to the subendocardial network (arrowheads in Fig. 7). These cells may represent a transitional form of Purkinje fibres at their junction with the working myocytes.
Discussion In the present investigation, we have demonstrated that IFAPa-400, a protein previously thought to be exclusively embryonic, is expressed in the mature vascular smooth muscle cells which also express vimentin. Using various antibodies, we have also demonstrated by immunoblot and immunohistological experiments that there is a topographical modulation of IFAPa-400 expression in the vascular smooth muscle cells of the great arteries. IFAPa-400 is abundant, as is vimentin, in the segments of the large arteries emerging from the heart but is progressively less abundant in the more distal segments. In contrast, desmin content gradually increases as the distance from the heart increases. Furthermore, this study demonstrates the presence of IFAPa-400 in the mature Purkinje fibres, a subset of cardiac myocytes specialize for the conduction of electrical impulses for the cardiac contractile process. Our observation that IFAPa-400 is present in the vascular smooth muscle cells of the adult chicken coincides with the expression of vimentin, which is also found in the vascular smooth muscle cells. In contrast, the IFS of other muscle cell types are composed pre-
et al dominantly of desmin. Previous studies have demonstrated that the vimentin content (or the vimentin to desmin ratio, depending on the species) decreases with the distance from the heart and has been suggested to be related to the elasticity of the arteries [8, 7, 13, 191. Whether these variations in the IF composition of smooth muscle cells reflect different mechanical requirements and/or differentiation processes remains an open and intriguing question. During myogenesis, vimentin is transiently expressed in skeletal, cardiac and visceral smooth muscle cells and is gradually replaced by desmin upon differentiation [2, 9, 24, 26, 211.We have recently shown that the transient expression of vimentin in non-differentiated muscle cells coincided with that of IFAPa-400, which co-localized and co-purified with vimentin [271. During in vivo or in vitro myogenesis, the expression of both vimentin and IFAPa-400 was shown to cease before terminal differentiation [q. The differences of IFAPa-400 and IF cornposition between proximal and distal segments of the arteries may reflect differences with respect to the requirements between the elastic and muscular components. Although the notion of elasticity is more readily conceived of as a physiological phenomenon rather than morphological, it is noteworthy that IFAPa-400 and vimentin are more abundant in the larger arteries closer to the heart. These vessels are assumed to act as conducting vessels and their elasticity is an important parameter in the maintenance of diastolic blood pressure and blood flow regulation. The presence of a ‘special’ IF cytoskeletal structure which includes the vimentin-IFAPa-400 combination in the smooth muscle cells of the elastic arteries may confer particular mechanical properties. With increasing distance from the heart, the IF cytoskeleton of the smooth muscle cells may then gradually adapt to meet more contractile functions, and change their vimentin and IFAPa-400 content for desmin and possibly an unknown accessory protein. The presence of IFAPa-400 in the Purkinje fibres could also be involved in the maintenance of their three-dimensional structure during the mechanical perturbations arising from the beating heart of could favour fast electrical conduction. These cells are known to
IFApa-400
in Vascular
contain specific intracellular filament components including significant amounts of IF proteins [22]. In birds, they have an elaborate IF cytoskeleton composed of desmin and vimentin [23, and our own observations]. Whether IFAPa-400 interacts with both vimentin and desmin remains to be determined but our previous observations have shown that both IFAPa-400 and desmin are co-localized in non-differentiated myoblasts [27, 61. The localization of ultrastructural IFAPa-400 at the junctions of intersecting IFS as well as its very high MW suggest that it may be a member of the family of proteins which have been described as cross-linker IFAPs [20]. One of these proteins, paranemin, has several properties similar to those of IFAPa-400. Both are chicken-specific and developmentally regulated. Paranemin is also down-regulated during differentiation of skeletal and visceral smooth muscle and the smooth muscle of the muscular arteries [15]. However, not only does paranemin remain in the smooth muscle cells of the elastic arteries and the Purkinje fibres, it also remains in mature ordinary myocardial cells, which is not the case for IFAPa-400. A further difference between IFAPa-400 and paranemin is the expression of the former in most, if not all, neuroectodermal cells prior to terminal differentiation [5] whereas the latter is only expressed in both embryonic and adult neural crest-derived Schwann cells [ 151. Moreover, the MW of IFAPa-400 estimated using our gel system is significantly higher than the values reported for paranemin (280 kD) [3]. In the type of gradient gel we have used (3-12 %), the apparent MW of IFAPa-400 is closer to 500 kD than to our previously reported value of 400kD [271. Titin (2.6 x 106), nebulin (600-800k), dystrophin (427k) and spectrin subunits (240 and 220k) were used as high MW reference markers. Although both IFAPa-400 and paranemin are IFAPs which may cross-link IFS, several of their characteristics appear distinct and it therefore remains to be determined if both proteins are related, either biochemically and/or at the gene level. The persistence of IFAPa-400 in some adult myocytes does not necessarily run counter to
Smooth
Muscle
881
the function that we have proposed for it during development [5, 61. In most myogenic neurogenic IFAPa-400 is and tissues, transiently co-expressed with vimentin during the structural re-organization of the IF- cytoskeleton accompanying cytodifferentiation. Assuming that IFAPa-400 is an IF cross-linker [6J, it may be either directly involved in the IF protein transition occurring in myogenic and neurogenic tissues, or alternatively, this protein may in some manner modulate cytoskeletal structures to accommodate the morphological re-arrangements imposed by development. Further characterization of IFAPa-400 may indicate that the extent of its molecular interactions may not be limited to IFS. The continued expression of IFAPa-400, together with vimentin, in some myogenic lineages such as proximal vascular smooth muscle cells and Purkinje fibres, may in fact reflect a cellular state more closely related to that of embryonic cells. Indeed, under certain circumstances, mature smooth muscle cells demonstrate the capability to re-enter the cell Together with their motile cycle [Xl. potential, another embryonic trait, this proliferative ability is a key event in the formation of atherosclerotic plaques [IA. It is thus conceivable that the embryonic-like IF cytoskeleton present in the smooth muscle cells of the elastic arteries and in the Purkinje fibres fulfills a specific structural need similar to that expressed by non-differentiated cells of the muscle and neural lineages.
Acknowledgements Supported in part by the MRC of Canada (grant PG-35 to M.V.) and by the Quebec Division of the Heart and Stroke Foundation of Canada (P.A.R. and M.V.). The authors are grateful to Yolande CBtC for her technical assistance, to Marie Duval, Jean-Luc Simard and Jean-Paul Valet for the preparation and validation of the antibodies and to Mary Ann Godbout for typing the manuscript. M .V . was a scholar from the Fonds de Ia Recherche en Sante du Quebec (FRSQ). R.W.C. was a visiting scientist at the Centre de Recherches du CHUL funded in part by the FRSQ.
M. viucemt
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1
BENNETT, G.S., 151-183.
Changes
in intermediate
4 5
6
7 8
9
10 11 12 13
14 15 16 17 18 19
20 21 22 23
24 25 26 27
composition
during
neurogenesis.
Curr
Top Dev
Biol 21,
(1987).
2 BENNETT, G. S. FELLINI, S., TOYAMA, 3
filament
et al
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23. 873-882 (1991)
Persistence of an Embryonic Intermediate Filament-associated in the Smooth Muscle Cells of Elastic Arteries and in Purkinje Michel
Vincent*,
CHUL
Sylvain
Levasseur,
R. William
Currief-
and Peter
Protein Fibres A. Rogers
Research Center and Department of Medicine, Lava1 University, Ste-Foy, Quk%ec, Canada (Received 14 August 1990, accepted in revisedform 7March 1991)
M. VINCENT, S. LEVASSEUR, R. W. CURRIE AND P. A. ROGERS. Persistence of an Embryonic Intermediate Filament-associated Protein in the Smooth Muscle Cells of Elastic Arteries and in Purkinje Fibres. Journal of Molecular and Cellular Cardiology (1991) 23, 873-882. During differentiation of most myogenic tissues, vimentin is transiently expressed as the intermediate flament (IF) protein subunit and is progressively replaced by desmin. However, smooth muscle cells of mature vascular tissue contain variable amounts of vimentin whose cellular content decreases as function of the distance from the heart. IFAPa-400 is a developmentally regulated IF crosslinker protein whose expression appears to parallel that of vimentin during chick myogenesis. Immunohistological and immunoblot techniques were employed to study the expression of this protein in cardiac and vascular smooth muscle cells of the adult chicken. As observed for vimentin, the expression of IFAPa-400 persists in the mature smooth muscle cells of the large arteries as they leave the heart. However, both of these proteins are downregulated according to a proximo-distal gradient with respect to the distance from the heart. Conversely, desmin is much more abundant in the distal segments of the aorta. Thus, the co-ordinate expression of vimentin with IFAPa-400 may be a characteristic feature of elastic vascular tissue in which it could meet mechanical requirements close to those of the embryonic cells expressing them. This hypothesis is supported by the observation that the single cell type which continues to express the vimentin-IFAPa-400 combination in the mature heart is the Purkinje fibres, which are also subjected to high mechanical tensions but in which myofibrils are generally sparse compared to working myocytes KEY WORDS: Vascular smooth filaments; Vimentin; IFAPa-400;
muscle cells; Elastic Desmin; Chicken.
Introduction Intermediate filaments (IF) constitute a class of cytoskeletal filaments which are morphologically and biochemically distinct from microfilaments and microtubules. The protein subunits of IF vary according to the cell type and this specificity led to the following system of classification: cytokeratins in epithelial cells, vimentin in mesenchymal cells, desmin in muscle cells, glial fibrillary acidic protein (GFAP) in glial cells and neurofilaments in neurones [12, 261. As the primary sequences for many IF proteins became available, a more precise classification into five distinct sequence types was proposed based on size and sequence differences in the central o-helical-rich rod domain whose conserved secondary structure accounts for the high degree of similarity in IF morphology [20]. *Author Boulevard I Present
to whom Laurier, address:
0022-2828/91/070873
arteries;
Atherosclerosis;
fibres;
Intermediate
Vimentin, desmin and GFAP share the IF type III sequence features. Desmin and GFAP are expressed in mature muscle cells and astrocytes respectively whereas vimentin, typically found in cells of mesenchymal origin, has also been reported in various cultured cells [26]. In addition, vimentin is known to be transiently expressed during muscle and nerve cell differentiation where it is gradually replaced by a second IF protein, characteristic of the mature cell type [9, 24, 26, I]. Vimentin is therefore unique in this respect since its expression appears to be regulated according to either celI or developmental type programme. We have recently identified and partially characterized a high molecular weight (MW) IF-associated protein, IFAPa-400 [21], whose expression is confined to the myogenic and the
correspondence should be addressed: Ontog6nese Ste-Foy, QuCbec, Canada GlV 4G2. Department of Anatomy, Dalhousie University, + 10$03.00/O
Purkinje
et GtnCtique Halifax,
Moltculaire Nova
Scotia,
CHUL,
2705
Canada.
0 1991 Academic
Press
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M. Vincent
neurogenic tissues of the chick embryo, i.e. the cell lineages that transiently express vimentin in development [5, s]. Due to this transient coexpression, we believe that IFAPa-400 is involved in the structural re-organization of the IF during muscle and neural cell differentiation. The vascular smooth muscle cells differ from the other myogenic lineages in that their IF may contain various amounts of vimentin in addition to desmin [8, 7,191. In mammals the ratio of vimentin to desmin may be related to the elasticity of large arteries. This ratio is higher in large arteries such as the aorta than in muscular arteries [ 7, 131. Since vimentin is expressed in some vascular smooth muscle cells and IFAPa-400 is transiently expressed during the developmental shift of vimentin to desmin in the other myogenic lineages, we decided to investigate whether this giant protein persists with vimentin in the elastic arteries. Here we report the persistence of IFAPa-400 in the vascular smooth muscle cells of the adult chicken; its expression is parallel to that of vimentin and the reversed of that of desmin. We also describe the persistence of IFAPa-400 in a cellular subpopulation of the mature heart, the IF-enriched Purkinje fibres.
Materials and Methods Six-month-old White Leghorn hens and fertilized eggs were purchased from a local hatchery. The eggs were maintained in a humidified forced-air incubator (80 to 90% relative humidity) at 38% and embryos were classified according to the number of incubation days.
Preparation
of antibodies
The monoclonal antibody (mab) F51H2 (IgM) was obtained by immunization of a Balb/c mouse with early differentiating chick embryo somites as described previously [271. The infilament-associated protein termediate (IFAPa-400) identified by this monoclonal was purified by ion exchange chromatography of embryonic heart ionic an high strength/detergent-resistant fraction solubilized in 8~ urea, 1OmM Tris-HCl, pH 7.4. The fractions containing IFAPa-400 reactivity were pooled, dialysed against 1OmM Tris-HCl, pH 7.4 and resolved by SDS-PAGE. A poly-
et al clonal antibody to IFAPa-400 was raised by injecting a rabbit with the IFAPa-400 band excised from acrylamide gels. The antibody was rendered monospecific by triple absorption of the serum on the protein immobilized on nitrocellulose. In order to validate our results, we used several monoclonals obtained after immunizing mice with IFAPa-400 prepared by ion exchange chromatography. Those from the IgG subclass showing immunofluorescence and immunoblot spatiotemporal patterns of reactivity identical to F51H2 were selected. A monoclonal antibody against vimentin (6G2) was generated using chicken erythrocyte vimentin as an antigen [5]. A rabbit polyclonal antiserum was raised against electrophoretically purified chicken desmin and has been previously characterized
WIGel electrophoresis and immunoblotting Hearts from embryonic and adult chickens were excised immediately following sacrifice, rinsed in PBS and frozen in liquid nitrogen. The aorta was dissected from the base of the heart to the abdominal bifurcation and further divided into proximal (ascending), intermediate (thoracic) and distal (abdominal) portions, then rinsed in PBS and frozen in liquid nitrogen. Frozen tissue samples were stored at - 80°C until electrophoretic analysis. Samples were homogenized in SDS sample buffer (2.3% SDS, 5% fimercaptoethanol, 10% glycerol, 0.062~ Tris pH 6.8) containing a cocktail of protease inhibitors (1mM phenylmethylsulphonyl fluoride, 5gglml leupeptin, 5pglml pepstatin, sag/ml antipain) and heated at 95°C for 3 min. Samples were then resolved by SDSPAGE on 3-12s or 7-15s gradient gels with an acrylamide: bis ratio of 30:0.15 in Laemmli’s buffer system [11]. After electrophoresis, the proteins were transferred electrophoretically to nitrocellulose at 200 mA overnight [25] and the MW markers visualized by staining with rouge Ponceau [4]. The nitrocellulose sheets were saturated with PBS containing 5% powdered milk [IO] before incubation with a primary antibody for lh at 37’C. Both ascitic fluid (mouse monoclonals F51H2 and 6G2) and rabbit antiserum were used at a dilution of 1: 1000. After extensive washes in PBS containing 0.05% Tween 20, the blots
875 were treated with either *251-labelled goat IgGs antimouse or antirabbit immunoglobulins. After washing, the blots wefe dried and exposed to Kodak X-Omat AR film at 80‘33 with intensifying screens.
Histologicalprocedures Ether-anaesthetized hens were perfused by cannulation of the left ventricle using heparinized PBS, 10% formalin and Perfix (Fisher Scientific) in sequence. The heart and the aorta were then dissected into small pieces and fixed overnight in Per-fix. After extensive washing in PBS, the pieces were progressively dehydrated in ethanol and toluene and embedded in a mixture of beeswax-Paraplast (1:8). Sections (5-7pm thick) were cut and mounted onto gelatin-coated glass slides. Before immunolabelling, the sections were deparaffinized in toluene, rehydrated through a decreasing ethanol series and washed several times in PBS.
Indirect immunoJuorescence Immunostaining on tissue sections was performed using monoclonal antibodies either as pure hybridoma supernatants or 1:50 dilution of ascitic fluids in PBS containing 0.5 % BSA and rabbit polyclonal antibodies at a dilution of 1:50. Sections were incubated in the primary antibody for 45 min at room temperature. After several PBS rinses, the sections were incubated for 45 min with FITCconjugated goat IgG antimouse immunoglobulins to detect monoclonal antibodies or with TRITC-conjugated goat IgG antirabbit immunoglobulins to reveal the polyclonal antibodies. In double-labelling experiments, primary antibodies (a monoclonal and a rabbit polyclonal) were mixed together before incubation and revealed as above. Mismatched incubations revealed no cross-reactivities between the two detection systems. Negative controls were routinely made by incubating the tissue sections either with irrelevant monoclonals or PBS containing 0.5% BSA. The specimens were mounted with p-phenylenediamine-PBS in glycerin [II] and observed under a Zeiss Axiophot microscope equipped with FITC and TRITC filter sets.
Results
Expressionof IFAPa-400 in adult aorticsmooth musclecells As part of our study of the developmental modulation of IFAPa-400 in chicken myogenic tissues, immunoblot experiments were carried out on whole homogenates from cardiac and aortic tissues explanted at different developmental states (Fig. 1). The expression of IFAPa-40 dramatically decreased during cardiogenesis as observed with skeletal and visceral smooth muscle. With equivalent amounts of proteins loaded on the gels, the 400 kD immunoreactive band was maximal at embryonic day (ED) 12, less intense at hatching (ED 21) and barely detectable in the adult. This pattern of immunoreactivity was completely reversed in the ascending aorta (Fig. 1). In this case, IFAPa-400 was hardly detectable in the first 10 or 11 days of incubation but was abundant in older embryos and in adult chickens. The same results (not shown) were obtained using the polyclonal antibody raised against IFAPa-400 and therefore 7
12
16
21
A
7
12
16
21
A
-240Kd - 220
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94
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Aorto
FIGURE 1. Expression of IFAPa-400 during heart and aorta embryogenesis. Immunoblot analysis of total tissue extracts from the heart and the ascending aorta prepared at different developmental stages and reacted with mab F51H2. Approximately equal amounts of proteins were loaded in each well. The expression of IFAPa-400 drastically decreased in the heart during development but increased in the aorta. The numbers represent the embryonic days. A, adult. The MW markers are shown in the margin.
876
FIGURE 2. Co-localization sections were double-labelled 6G2 respectively. IFAPa-400 the vimentin-rich connective (185 x )
M. Vincent
et al
of IFAPa-400 and vimentin in the smooth muscle cells ofthe ascending aorta. Transverse for IFAPa-400 (a) and vimentin (b) using the affinity-purified polyclonal antibody and mab co-localized with vimentin in the smooth muscle cells (bright layers) but was absent from tissue cells [in~l&~&~r punctatc staining in (b)] and from the endothelial cells (arrows).
demonstrated that IFAPa-400 down-regulation observed during myogenesis and neurogenesis does not occur during aorta ontogenesis. Since IFAPa-400 expression appeared closely related to that of vimentin during ontogenesis, we investigated whether its distribution in a mature tissue coincided with that of vimentin. Using double immunofluorescence, IFAPa-400 was shown to co-localize with vimentin in the smooth muscle cells of the ascending aorta but was absent from the vimentin-positive fibroblast-like cells dispersed in the interlamellar connective tissue layers (Fig. 2). In this large vessel, vimentin was also shown to be enriched in endothelial cells but no IFAPa-400 could be detected in this cell type (arrows in Fig. 2). It appears therefore that IFAPa-400 is not an obligatory companion of vimentin and that its expression in the adult aorta is confined to the smooth muscle cells.
Topopafihic
modulation of IFAPa-400 expression in the aorta
and IF
Three contiguous segments of the aorta and the pulmonary artery were examined by immunoblot to determine whether gradients of expression of IFS occurred along the length of the avian large arteries (Fig. 3) The segment of the aorta most proximal to the heart was excised just above the aortic valve and included the aortic arch. The intermediate portion corresponded to the aorta thoratalis whereas the most distal part comprised the beginning of the aorta abdominalis down to the renal arteries. The proximal portion of the pulmonary artery consisted of the common trunk originating from the right ventricle, the intermediate portion included the right and left pulmonary division and the fraction corresponding to the most distal segment included the intrapulmonary ramifications which we carefully dissected free from adjacent tissues.
877
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- 116 -64 -66 - 42
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- 42
FIGURE 3. Variations of IFAPa-400 and IF expression along the aorta and the pulmonary artery. Immunoblot analysis of total tissue extracts prepared from three contiguous segments of the aorta and the pulmonary artery and reacted with mabs F5lH2 and 6G2 and with the antidesmin serum. In each case, the extracts from the heart most proximal segments arc on the left lane (pmx). Compared to the typical IF composition of the mature muscle cell in which desmin (ties) was abundant and vimentin (vim) and IFAPa-400 were faint (heart, first lane), the segments of both arteries most proximal to the heart contained high concentrations of IFAPa-400 and vimentin and little desmin. The most distal segments were enriched with desmin and contained little or no vimentin and IFAPa-400. The MW markets ate shown in the margin. Rat skeletal muscle extracts were run in the top gel to provide additional high MW markers for IFAPa-400 MW determination. (titin z 226OkD; nebulin 6008OOkD)
vimentin-rich most proximal parts (Fig. 3). Thus the IF composition of the muscular wall of the large arteries coming from the heart undergoes rapid changes along their length. Those portions closer to the heart contain predominantly vimentin which presumably is associatedwith IFAPa-400 and trace amounts of desmin whereasthe most distal segmentsare more representative of the other mature muscle tissuesin that they are enriched with desmin but contain little or no vimentin and IFAPa-400. This typical IF composition of the mature muscle can be visualized in the heart (Fig. 3, first lane) in which desmin is abundant while IFAPa-400 and vimentin are barely detectable (seebelow). To verify whether the quantitative changes in the aorta IF composition arose from variations in the number of cells expressing these proteins, immunofluorescence microscopy was performed on aortic transverse sections at different levels. As shown above (see Fig. 2), IFAPa-400 and vimentin co-localized in the smooth muscle cellsof the proximal part of the aorta. At the level of the iliac bifurcation, however, double-labellings using the afhnitypurified rabbit antiserum to IFAPa-400 and the monoclonal 6G2 antivimentin revealed that both proteins were much lessabundant in the smooth muscle cells (Fig. 4). Also consistent with the immunoblot pattern, the desmin fluorescence was strong [Fig. 5(b)]. Although vimentin reactivity was weak in the smooth muscle cells, sporadic staining was observed either in the endothelial cells [arrow in Fig. 4(a)] or in putative mesenchymal components [Fig. 5(a)]. IFAPa-400
and vimmtin
in heart PurkinjeJibres
Immunoblot analysis of adult cardiac tissue using various IFAPa-400 antibodies consistently showed a faint immunoreactive band at the apparent molecular weight corresponding to IFAPa-400 ( see first lane in Fig. 3). Extreme care was taken to avoid any contamination by the outcoming arteries. Immunofluorescence staining of adult heart sections demonstrated the absence of In both arteries, parallel immunoblot ex- IFAPa-400 immunoreactivity in ordinary periments using F51H2, monoclonal6G2 anti- myocardial cells but a very high level in a vimentin and the antidesmin serum revealed subset of cells primarily located around blood opposite gradients for vimentin and desmin vessels (Fig. 6) and underneath the and high concentrations of IFAPa-400 in their endocardium (arrows in Fig. 7). These large
FIGURE 4. Absence of IFAPa-400 aorta at the level of the lilac bifurcation proteins was very faint in the smooth (arrow). (340 x )
and vimentin in the smooth muscle cells of distal aorta. Transverse sections of ‘the were double-labelled for vimentin (a) and IFAPa-400 (b). The staining for 1:10th muscle cells but some vimentin reactivity could be seen in the endothelial s:ells
FIGURE 5. Expression of desmin in the smooth muscle cells of the distal aorta. Transvene sections of the aorta at the level of the iliac bifurcation were double-labelled for vimentin (a) and desmin (b). The smooth muscle cells exhibit st rong desmin fluorescence. Vimentin reactivity is confined to some putative mesenchymal components. (350 x )
IFApa-400
in Vascular
Smooth
Muscle
heart FI( 3URE 6. Expression of IFAPa-400 in the Purkinje fibres of the mature heart. Sections of the adult chicken labelled for IFAPa-400 with the monoclonal antibody F51H2. Strong fluorescence was observed in pu tative cells. clustered around a blood vessel. Note the absence of reactivity in the surrounding myocardial Purki ln,je fibres (500 X)
FI’ were unde cells, (490
GURE labelled ,meath which X)
7. Expression for IFAPa-400 the endocardium may represent
I heart of IFAPa-400 in the Purkinje fibres of the mature heart. Sections of the adult thicker with the monoclonal antibody F51H2. Purkinje fibres forming one-cell thick s trands were intensely labelled (arrows). Some fluorescence was also observed in the surrou mding leads). a transitional form between Purkinje fibres and working cardiac myocytes (arrowh
880
M. Vincent
cells have centrally located nuclei and occasionally a few myofibrils can be observed arranged peripherally around central refringent cytoplasmic material. These cells have been previously identified in the chicken heart as Purkinje fibres [18]. When labelled with desmin antibody, these Purkinje fibres were more fluorescent than the surrounding myocytes (data not shown). Double-labelling heart sections with polyclonal anti-IFAPa-400 and monoclonal antivimentin demonstrated the coincidence of both proteins in the Purkinje fibre cytoplasm. In contrast, other vimentin-positive cells including endothelial cells and fibroblasts were devoid of IFAPa-400 (data not shown). Although IFAPa-400 could not be detected in ordinary myocytes, some staining was occasionally observed in subsets of cells usually close to the subendocardial network (arrowheads in Fig. 7). These cells may represent a transitional form of Purkinje fibres at their junction with the working myocytes.
Discussion In the present investigation, we have demonstrated that IFAPa-400, a protein previously thought to be exclusively embryonic, is expressed in the mature vascular smooth muscle cells which also express vimentin. Using various antibodies, we have also demonstrated by immunoblot and immunohistological experiments that there is a topographical modulation of IFAPa-400 expression in the vascular smooth muscle cells of the great arteries. IFAPa-400 is abundant, as is vimentin, in the segments of the large arteries emerging from the heart but is progressively less abundant in the more distal segments. In contrast, desmin content gradually increases as the distance from the heart increases. Furthermore, this study demonstrates the presence of IFAPa-400 in the mature Purkinje fibres, a subset of cardiac myocytes specialize for the conduction of electrical impulses for the cardiac contractile process. Our observation that IFAPa-400 is present in the vascular smooth muscle cells of the adult chicken coincides with the expression of vimentin, which is also found in the vascular smooth muscle cells. In contrast, the IFS of other muscle cell types are composed pre-
et al dominantly of desmin. Previous studies have demonstrated that the vimentin content (or the vimentin to desmin ratio, depending on the species) decreases with the distance from the heart and has been suggested to be related to the elasticity of the arteries [8, 7, 13, 191. Whether these variations in the IF composition of smooth muscle cells reflect different mechanical requirements and/or differentiation processes remains an open and intriguing question. During myogenesis, vimentin is transiently expressed in skeletal, cardiac and visceral smooth muscle cells and is gradually replaced by desmin upon differentiation [2, 9, 24, 26, 211.We have recently shown that the transient expression of vimentin in non-differentiated muscle cells coincided with that of IFAPa-400, which co-localized and co-purified with vimentin [271. During in vivo or in vitro myogenesis, the expression of both vimentin and IFAPa-400 was shown to cease before terminal differentiation [q. The differences of IFAPa-400 and IF cornposition between proximal and distal segments of the arteries may reflect differences with respect to the requirements between the elastic and muscular components. Although the notion of elasticity is more readily conceived of as a physiological phenomenon rather than morphological, it is noteworthy that IFAPa-400 and vimentin are more abundant in the larger arteries closer to the heart. These vessels are assumed to act as conducting vessels and their elasticity is an important parameter in the maintenance of diastolic blood pressure and blood flow regulation. The presence of a ‘special’ IF cytoskeletal structure which includes the vimentin-IFAPa-400 combination in the smooth muscle cells of the elastic arteries may confer particular mechanical properties. With increasing distance from the heart, the IF cytoskeleton of the smooth muscle cells may then gradually adapt to meet more contractile functions, and change their vimentin and IFAPa-400 content for desmin and possibly an unknown accessory protein. The presence of IFAPa-400 in the Purkinje fibres could also be involved in the maintenance of their three-dimensional structure during the mechanical perturbations arising from the beating heart of could favour fast electrical conduction. These cells are known to
IFApa-400
in Vascular
contain specific intracellular filament components including significant amounts of IF proteins [22]. In birds, they have an elaborate IF cytoskeleton composed of desmin and vimentin [23, and our own observations]. Whether IFAPa-400 interacts with both vimentin and desmin remains to be determined but our previous observations have shown that both IFAPa-400 and desmin are co-localized in non-differentiated myoblasts [27, 61. The localization of ultrastructural IFAPa-400 at the junctions of intersecting IFS as well as its very high MW suggest that it may be a member of the family of proteins which have been described as cross-linker IFAPs [20]. One of these proteins, paranemin, has several properties similar to those of IFAPa-400. Both are chicken-specific and developmentally regulated. Paranemin is also down-regulated during differentiation of skeletal and visceral smooth muscle and the smooth muscle of the muscular arteries [15]. However, not only does paranemin remain in the smooth muscle cells of the elastic arteries and the Purkinje fibres, it also remains in mature ordinary myocardial cells, which is not the case for IFAPa-400. A further difference between IFAPa-400 and paranemin is the expression of the former in most, if not all, neuroectodermal cells prior to terminal differentiation [5] whereas the latter is only expressed in both embryonic and adult neural crest-derived Schwann cells [ 151. Moreover, the MW of IFAPa-400 estimated using our gel system is significantly higher than the values reported for paranemin (280 kD) [3]. In the type of gradient gel we have used (3-12 %), the apparent MW of IFAPa-400 is closer to 500 kD than to our previously reported value of 400kD [271. Titin (2.6 x 106), nebulin (600-800k), dystrophin (427k) and spectrin subunits (240 and 220k) were used as high MW reference markers. Although both IFAPa-400 and paranemin are IFAPs which may cross-link IFS, several of their characteristics appear distinct and it therefore remains to be determined if both proteins are related, either biochemically and/or at the gene level. The persistence of IFAPa-400 in some adult myocytes does not necessarily run counter to
Smooth
Muscle
881
the function that we have proposed for it during development [5, 61. In most myogenic neurogenic IFAPa-400 is and tissues, transiently co-expressed with vimentin during the structural re-organization of the IF- cytoskeleton accompanying cytodifferentiation. Assuming that IFAPa-400 is an IF cross-linker [6J, it may be either directly involved in the IF protein transition occurring in myogenic and neurogenic tissues, or alternatively, this protein may in some manner modulate cytoskeletal structures to accommodate the morphological re-arrangements imposed by development. Further characterization of IFAPa-400 may indicate that the extent of its molecular interactions may not be limited to IFS. The continued expression of IFAPa-400, together with vimentin, in some myogenic lineages such as proximal vascular smooth muscle cells and Purkinje fibres, may in fact reflect a cellular state more closely related to that of embryonic cells. Indeed, under certain circumstances, mature smooth muscle cells demonstrate the capability to re-enter the cell Together with their motile cycle [Xl. potential, another embryonic trait, this proliferative ability is a key event in the formation of atherosclerotic plaques [IA. It is thus conceivable that the embryonic-like IF cytoskeleton present in the smooth muscle cells of the elastic arteries and in the Purkinje fibres fulfills a specific structural need similar to that expressed by non-differentiated cells of the muscle and neural lineages.
Acknowledgements Supported in part by the MRC of Canada (grant PG-35 to M.V.) and by the Quebec Division of the Heart and Stroke Foundation of Canada (P.A.R. and M.V.). The authors are grateful to Yolande CBtC for her technical assistance, to Marie Duval, Jean-Luc Simard and Jean-Paul Valet for the preparation and validation of the antibodies and to Mary Ann Godbout for typing the manuscript. M .V . was a scholar from the Fonds de Ia Recherche en Sante du Quebec (FRSQ). R.W.C. was a visiting scientist at the Centre de Recherches du CHUL funded in part by the FRSQ.
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1
BENNETT, G.S., 151-183.
Changes
in intermediate
4 5
6
7 8
9
10 11 12 13
14 15 16 17 18 19
20 21 22 23
24 25 26 27
composition
during
neurogenesis.
Curr
Top Dev
Biol 21,
(1987).
2 BENNETT, G. S. FELLINI, S., TOYAMA, 3
filament
et al
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