RAPID COMMUNICATION
ZIPGRAM
INTERMEDIATE FILAMENTS CONNECT Z-DISCS IN ADULT CHICKEN MUSCLE MAUREEN PRICE AND JOSEPH W. SANGER Department of Anatomy, School of Medicine/G3, University of Pennsylvania , Phi 1 adel phia , Pennsylvania 19104 ABSTRACT When adult chicken skeletal myofibrils are treated with a myosin-extracting solution, the Z-discs with attached actin filaments retain their linear connections with one another in the extracted myofibril. The sarcomere length increases in the extracted myofibrils from a control length of 2.5 pm up to 6 pm. In a sarcomere, eight to fifty 10 nm filaments can be seen in parallel array in the H-zone. The 10 nm-wide filaments do not bind heavy meromyosin and are two to four pm in length. These intermediate filaments are postulated to be an integral part of the sarcomere, connecting Z-bands along the length of the myofibril. Filaments, 10 nm in diameter, are present in nearly all cells (Ishikawa et al. , '68; Weihing, '79).
In one of the first cell types in which these
filaments were demonstrated, embryonic myotubes, they were shown to be intermediate in size between actin and myosin filaments, and the term intermediate filament was introduced to describe these and all other cytoplasmic filaments with diameters in the range of 10 nm (Ishikawa et al., '68). The role of these intermediate filaments i s not yet known. Although intermediate filaments fill the space between the developing myofibrils of embryonic myotubes (Ishikawa et al., '68;Kelly, '69; Fischman, '72) and embryonic chick cardiac muscle cells (Rash et al., '70) and are abundant in both embryonic (Sanger et a1 ., '78) and adult smooth muscle (Somlyo et al., ' 7 6 ) , they have not been reported in adult skeletal muscle cells. We have treated adult chick myofibrils with solutions which extract
263
myosin and have found t h a t when the thick filaments a r e removed, intermediate filaments can be seen within t he sarcomeres of t h e muscle.
We believe t h a t
these filaments connect the Z-discs together within a myofibril. MATERIALS AND METHODS
Myofibrils were obtained by homogenizing s t r i p s
of ad u l t chicken bre a st muscle t h a t had been glycerinated (50% glycerol and 50% standard s a l t sol uti on (0.1 M K C 1 , 0.01 M PO4 buf fer , -.001 M MgC12).
Membranes were sol ubil iz e d by washing the myofibrils several times with a solution of Triton-X 100 (1%)in standard s a l t . placed in low s a l t .
The myofibrils were then
Myosin was extracted from t h e myofibrils in an ice-cold
solution of 0 . 1 M K4P207, l O m M MgC12, 0.5 M KC1, pH 7.0 (Hasselbach and Schneider, '51; Go11 e t a l . , ' 7 7 ) .
Following myosin e x t r a c t i o n , the myofibrils
were washed and resuspended in low s a l t medium. t o s t i c k t o t h e gl a ss walls of the tubes.
Extracted myofibrils tended
This was overcome by maintaining
the extracted myofibrils in a pyrophosphate solution ( 0 . 1 M K4P207, 10 rnM MgC12, pH 7 . 0 ) .
Control myofibrils and myosin-extracted myofibrils were
placed on carbon-coated grids and allowed t o s e t t l e f o r t h i r t y seconds.
The
attached rnyofibrils were washed with several drops of 0 . 1 M KC1 in order t o remove pyrophosphate s a l t s which i n t e r f e r e with the negative s t a i n i n g process. Some g r i d s were then incubated with r a b b i t heavy meromyosin (HMM) (2. 5 mg/ml) which had been prepared by t h e method of Szent-Gyorgyi ( ' 5 3 ) . f i b r i l s were then washed again with 0 . 1 M KC1.
These myo-
B o t h HMM-stained and control
extracted g r i d s were stained with uranyl a c e t a t e .
Dispersed actin and myosin
filaments from Triton-X t re a t e d chicken myofibrils were a l s o prepared using the ATP relaxing solution of Huxley ( ' 6 3 ) .
Drops of t h e homogenate containing
the t h i n and thick filaments were placed on carbon-coated g r i d s and negatively stained with uranyl a c e t a t e .
RESULTS
In control myofibrils negatively stained on carbon-coated grids
264
no signs of intermediate filaments were observed (fig. 1).
Myosin-extracted
myofibril s retained their integrity during repeated washes even though no A-band material could be observed in the light microscope. These myosinextracted myofibrils were then examined in the electron microscope after negatively staining them. Filamentous actin remained attached to either side of the Z-discs (fig. 2).
The actin filaments were 1 micron long in both
control and myosin-extracted myofibrils. The Z-discs in the extracted myofibrils were often equidistant apart within one myofibril. they were five microns apart (fig. 2).
In some myofibrils
In the H-zone between the Z-discs
with attached actin filaments was a small number of filaments averaging 10 nm in diameter (60 were measured).
Ninety-three percent o f the filaments had
diameters between 93 and 130 8. The adult chicken myofibrils have an average width of about 2
pm.
There was a range of eight to fifty parallel fi aments
in each sarcomere. Individual filaments were at least 2 um long, and some could be traced for 2 to 6 pm between the tips of oppositely opposed x t i n filaments (fig. 3 ) .
None o f these 10 nm filaments possessed periodicities
which could be observed with negative staining. When heavy meromyosin was added to myosin-extracted myofibrils, arrowheaded complexes were found on the actin filaments but not in the 10 nm filaments connecting the serial Z-bands and actin filaments. In addition to the numerous actin and myosin filaments released intact when fibrils were homogenized in ATP relaxing solution, there was a small number of filaments 10 nm in diameter. These 10 nm filaments were two to four times longer than the actin (6 nm in diameter) filaments, which were almost without exception 1 pm in length. The rod-shaped myosin filaments (18 nm in diameter) were about 1.6 um in length and were further distinguished
from both the actin and 10 nm filaments by the presence of cross-bridges.
265
DISCUSSION Huxley and Hanson ('54)postulated that the basis for contraction in skeletal muscle was the sliding of thin and thick filaments past one another. In the same paper and a preceding one by Hanson and Huxley ('53) they reported that although myosin could be extracted from the myofibrils, the Z-discs kept their relationship to one another. Hanson and Huxley ('55) suggested that a system of filaments (S filaments) connected the Z-bands together in a myofibril. Later the evidence was re-examined and the existence of a third filament system was called into doubt by Huxley ('68). Since that time a number of papers have appeared which have suggested that a third type of filament is present in skeletal muscle, but the evidence has not been very convincing for vertebrate muscles. (See review of references by dos Remedios and Gilmour, '78 and by Granger and Lazarides, '78). Our results indicate that myosin-extracted myofibrils retain their 1 inear integrity while in suspension. These myosin-extracted myofibrils can be repeatedly spun down in a centrifuge and resuspended. Previously, workers have extracted myofibrils while they were attached to glass (for the light microscope) or to coated grids (for the electron microscope) and thus their observations have been subject to the criticism that protein extraction was incomplete or that the Z-bands retained their disposition because they were stuck to a surface (Granger and Lazarides, '78). Our negatively stained
FIGURE LEGENDS 1 Control chicken myofibril negatively stained on grid with uranyl acetate. Small arrows point to Z-discs and big arrows to H-zone. X 9300. 2 Myofibril from which myosin has been removed. Note the connect ng intermediate filaments in the H-zone (arrows). X 3000. 3 A myosin-extracted myofibril that has been decorated with heavy meromyosin (HMM). The long interconnecting filaments (large arrows) do not bind HMM and thus are smooth. The actin filamen S bind HMM (small arrows). X 22,000.
266
preparations of extracted myofibrils reveal that the A-bands have been removed. The adjacent Z-discs seem to be connected by aperiodic filaments having a diameter of about 10 nm and a length of u p to 5 urn.
These connecting fila-
ments are thicker than actin and do not bind heavy meromyosin. Thus, they are not actin. The length, diameter, insolubility in high salt and lack o f cross bridges indicate that these filaments are not myosin. We feel that these 10 nm connecting filaments are the same as the intermediate filaments found in embryonic muscle.
The experiments involving extraction and centrifugation demonstrate that the connecting intermediate filaments are tightly associated with the complexes of Z-bands and actin. In smooth muscle intermediate filaments are closely associated with the periphery o f the dense bodies which are the equivalent of Z-bands (Somlyo et al. , ‘76). It still remains to be determined where the intermediate filaments are attached in the skeletal myofibrils, but we expect that future work will demonstrate that they are anchored at the periphery of the Z-discs. The location of the intermediate filaments on the periphery of the Z-discs would ensure free movement of the interacting thin and thick filaments. ACKNOWLEDGMENTS The authors are grateful to Dr. Jean M. Sanger for her critical and constructive reading of this manuscript. Study supported by grants from the National Institutes of Health (GM25653 to J.W.S. and HL-15835 to the Pennsylvania Muscle Institute).
M.
P.
was supported by an Anatomy Training Grant (GM-00281). LITERATURE CITED dos Remedios, C. G., and D. Gilmour 1978 Is there a third type of filament in striated muscles? J. Biochem., 84: 235-238. Fischman, D. A. 1972 Development o f striated muscle. In: The Structure and Function of Muscle, Vol. 1, G. H. Boune, ed., Academic Press, New York, 2nd Ed., pp. 75-148. 268
Goll, D. E . , M. H . Stromer, R. M. Robson, B. M. Luke and K. S. Hammond 1977 E x t r a c t i o n , p u r i f i c a t i o n and l o c a l i z a t i o n of a - a c t i n i n from asynchromous i n s e c t f l i g h t muscle. I n : I n s e c t F l i g h t Muscle, R . T . Tregear, ed. North Holland Publ. Co., Amsterdam, p p . 15-40. Granger, B . L . , and E . Lazarides 1978 The e x i s t e n c e o f an i n s o l u b l e Z-disc s c a f f o l d i n chicken s k e l e t a l muscle. Cell , 15: 1253-1268. Hanson, J . , and H . E . Huxley 1953 S t r u c t u r a l b a s i s of t h e c r o s s s t r i a t i o n s in muscle. Nature, 1 7 2 : 530-532. Hanson, J . , and H . E. Huxley 1955 The s t r u c t u r a l b a s i s o f c o n t r a c t i o n i n s t r i a t e d muscle. Sym. SOC. Exper. Biology, 9: 228-264. Hasselbach, W., and G . Schneider 1951 Der L-Myosin-und Aktingehalt des Kaninchenmuskels. Biochem. Z . , 321: 461-475. Huxley, H . E. 1963 Electron microscopic studies on the s t r u c t u r e of natural and s y n t h e t i c f i l a m e n t s from s t r i a t e d muscle. J . Mol. B i o l . , 7: 281-307. Huxley, H . E. 1968 I n : Symposium on Muscle. Symp. b i o l . H u n g . , 8: 249-250. Huxley, H . E . , and Hanson, J . 1954 Changes i n the c r o s s - s t r i a t i o n of muscle d u r i n g c o n t r a c t i o n and s t r e t c h and t h e i r s t r u c t u r a l i n t e r p r e t a t i o n . Nature, 173: 973-976. Ishikawa, H . , R . Bischoff and H . Holtzer 1968 Mitosis and intermediate. s i z e d f i l a m e n t s in developing muscle. 3 . C e l l . B i o l . , 38: 538-555. Kelly, D . E . 1969 Myofibrillogenesis and Z-band d i f f e r e n t i a t i o n . Anat. Rec., 163: 403-426. Rash, J . E . , J . J . Biesele and G. 0. Gey 1970 Three c l a s s e s of f i l a ments i n c a r d i a c d i f f e r e n t i a t i o n . J . U l t r a s t r u c t . Res., 33: 408-435. Sanger, J . W., J . M. Sanger and J . G w i n n 1978 Analysis o f r o l e s o f a c t i n , microtubules and intermediate f i l a m e n t s i n spreading f i b r o b l a s t s and muscle c e l l s . J . Cell B i o l . , 79: 272a. Somlyo, A . P . , A. V. Somlyo, F. T . Ashton and J . V a l l i e r e s 1976 Vertebrate and smooth muscle: ul t r a s t r u c t u r e and f u n c t i o n . Cold Spring Harbor Conf. Cell P r o l i f . , 3: 165-183. Szent-Gyorgyi, A. G . 1953 Meromyosins, t h e subunits o f myosin. Arch Biochem Biophys., 42: 305-320. Weihing, R . R. 1979 The cytoskeleton and plasma membrane. Meth. Archive. exp. P a t h o l . , 8: 42-109.
269
ZIPGRAM
INTERMEDIATE FILAMENTS CONNECT Z-DISCS IN ADULT CHICKEN MUSCLE MAUREEN PRICE AND JOSEPH W. SANGER Department of Anatomy, School of Medicine/G3, University of Pennsylvania , Phi 1 adel phia , Pennsylvania 19104 ABSTRACT When adult chicken skeletal myofibrils are treated with a myosin-extracting solution, the Z-discs with attached actin filaments retain their linear connections with one another in the extracted myofibril. The sarcomere length increases in the extracted myofibrils from a control length of 2.5 pm up to 6 pm. In a sarcomere, eight to fifty 10 nm filaments can be seen in parallel array in the H-zone. The 10 nm-wide filaments do not bind heavy meromyosin and are two to four pm in length. These intermediate filaments are postulated to be an integral part of the sarcomere, connecting Z-bands along the length of the myofibril. Filaments, 10 nm in diameter, are present in nearly all cells (Ishikawa et al. , '68; Weihing, '79).
In one of the first cell types in which these
filaments were demonstrated, embryonic myotubes, they were shown to be intermediate in size between actin and myosin filaments, and the term intermediate filament was introduced to describe these and all other cytoplasmic filaments with diameters in the range of 10 nm (Ishikawa et al., '68). The role of these intermediate filaments i s not yet known. Although intermediate filaments fill the space between the developing myofibrils of embryonic myotubes (Ishikawa et al., '68;Kelly, '69; Fischman, '72) and embryonic chick cardiac muscle cells (Rash et al., '70) and are abundant in both embryonic (Sanger et a1 ., '78) and adult smooth muscle (Somlyo et al., ' 7 6 ) , they have not been reported in adult skeletal muscle cells. We have treated adult chick myofibrils with solutions which extract
263
myosin and have found t h a t when the thick filaments a r e removed, intermediate filaments can be seen within t he sarcomeres of t h e muscle.
We believe t h a t
these filaments connect the Z-discs together within a myofibril. MATERIALS AND METHODS
Myofibrils were obtained by homogenizing s t r i p s
of ad u l t chicken bre a st muscle t h a t had been glycerinated (50% glycerol and 50% standard s a l t sol uti on (0.1 M K C 1 , 0.01 M PO4 buf fer , -.001 M MgC12).
Membranes were sol ubil iz e d by washing the myofibrils several times with a solution of Triton-X 100 (1%)in standard s a l t . placed in low s a l t .
The myofibrils were then
Myosin was extracted from t h e myofibrils in an ice-cold
solution of 0 . 1 M K4P207, l O m M MgC12, 0.5 M KC1, pH 7.0 (Hasselbach and Schneider, '51; Go11 e t a l . , ' 7 7 ) .
Following myosin e x t r a c t i o n , the myofibrils
were washed and resuspended in low s a l t medium. t o s t i c k t o t h e gl a ss walls of the tubes.
Extracted myofibrils tended
This was overcome by maintaining
the extracted myofibrils in a pyrophosphate solution ( 0 . 1 M K4P207, 10 rnM MgC12, pH 7 . 0 ) .
Control myofibrils and myosin-extracted myofibrils were
placed on carbon-coated grids and allowed t o s e t t l e f o r t h i r t y seconds.
The
attached rnyofibrils were washed with several drops of 0 . 1 M KC1 in order t o remove pyrophosphate s a l t s which i n t e r f e r e with the negative s t a i n i n g process. Some g r i d s were then incubated with r a b b i t heavy meromyosin (HMM) (2. 5 mg/ml) which had been prepared by t h e method of Szent-Gyorgyi ( ' 5 3 ) . f i b r i l s were then washed again with 0 . 1 M KC1.
These myo-
B o t h HMM-stained and control
extracted g r i d s were stained with uranyl a c e t a t e .
Dispersed actin and myosin
filaments from Triton-X t re a t e d chicken myofibrils were a l s o prepared using the ATP relaxing solution of Huxley ( ' 6 3 ) .
Drops of t h e homogenate containing
the t h i n and thick filaments were placed on carbon-coated g r i d s and negatively stained with uranyl a c e t a t e .
RESULTS
In control myofibrils negatively stained on carbon-coated grids
264
no signs of intermediate filaments were observed (fig. 1).
Myosin-extracted
myofibril s retained their integrity during repeated washes even though no A-band material could be observed in the light microscope. These myosinextracted myofibrils were then examined in the electron microscope after negatively staining them. Filamentous actin remained attached to either side of the Z-discs (fig. 2).
The actin filaments were 1 micron long in both
control and myosin-extracted myofibrils. The Z-discs in the extracted myofibrils were often equidistant apart within one myofibril. they were five microns apart (fig. 2).
In some myofibrils
In the H-zone between the Z-discs
with attached actin filaments was a small number of filaments averaging 10 nm in diameter (60 were measured).
Ninety-three percent o f the filaments had
diameters between 93 and 130 8. The adult chicken myofibrils have an average width of about 2
pm.
There was a range of eight to fifty parallel fi aments
in each sarcomere. Individual filaments were at least 2 um long, and some could be traced for 2 to 6 pm between the tips of oppositely opposed x t i n filaments (fig. 3 ) .
None o f these 10 nm filaments possessed periodicities
which could be observed with negative staining. When heavy meromyosin was added to myosin-extracted myofibrils, arrowheaded complexes were found on the actin filaments but not in the 10 nm filaments connecting the serial Z-bands and actin filaments. In addition to the numerous actin and myosin filaments released intact when fibrils were homogenized in ATP relaxing solution, there was a small number of filaments 10 nm in diameter. These 10 nm filaments were two to four times longer than the actin (6 nm in diameter) filaments, which were almost without exception 1 pm in length. The rod-shaped myosin filaments (18 nm in diameter) were about 1.6 um in length and were further distinguished
from both the actin and 10 nm filaments by the presence of cross-bridges.
265
DISCUSSION Huxley and Hanson ('54)postulated that the basis for contraction in skeletal muscle was the sliding of thin and thick filaments past one another. In the same paper and a preceding one by Hanson and Huxley ('53) they reported that although myosin could be extracted from the myofibrils, the Z-discs kept their relationship to one another. Hanson and Huxley ('55) suggested that a system of filaments (S filaments) connected the Z-bands together in a myofibril. Later the evidence was re-examined and the existence of a third filament system was called into doubt by Huxley ('68). Since that time a number of papers have appeared which have suggested that a third type of filament is present in skeletal muscle, but the evidence has not been very convincing for vertebrate muscles. (See review of references by dos Remedios and Gilmour, '78 and by Granger and Lazarides, '78). Our results indicate that myosin-extracted myofibrils retain their 1 inear integrity while in suspension. These myosin-extracted myofibrils can be repeatedly spun down in a centrifuge and resuspended. Previously, workers have extracted myofibrils while they were attached to glass (for the light microscope) or to coated grids (for the electron microscope) and thus their observations have been subject to the criticism that protein extraction was incomplete or that the Z-bands retained their disposition because they were stuck to a surface (Granger and Lazarides, '78). Our negatively stained
FIGURE LEGENDS 1 Control chicken myofibril negatively stained on grid with uranyl acetate. Small arrows point to Z-discs and big arrows to H-zone. X 9300. 2 Myofibril from which myosin has been removed. Note the connect ng intermediate filaments in the H-zone (arrows). X 3000. 3 A myosin-extracted myofibril that has been decorated with heavy meromyosin (HMM). The long interconnecting filaments (large arrows) do not bind HMM and thus are smooth. The actin filamen S bind HMM (small arrows). X 22,000.
266
preparations of extracted myofibrils reveal that the A-bands have been removed. The adjacent Z-discs seem to be connected by aperiodic filaments having a diameter of about 10 nm and a length of u p to 5 urn.
These connecting fila-
ments are thicker than actin and do not bind heavy meromyosin. Thus, they are not actin. The length, diameter, insolubility in high salt and lack o f cross bridges indicate that these filaments are not myosin. We feel that these 10 nm connecting filaments are the same as the intermediate filaments found in embryonic muscle.
The experiments involving extraction and centrifugation demonstrate that the connecting intermediate filaments are tightly associated with the complexes of Z-bands and actin. In smooth muscle intermediate filaments are closely associated with the periphery o f the dense bodies which are the equivalent of Z-bands (Somlyo et al. , ‘76). It still remains to be determined where the intermediate filaments are attached in the skeletal myofibrils, but we expect that future work will demonstrate that they are anchored at the periphery of the Z-discs. The location of the intermediate filaments on the periphery of the Z-discs would ensure free movement of the interacting thin and thick filaments. ACKNOWLEDGMENTS The authors are grateful to Dr. Jean M. Sanger for her critical and constructive reading of this manuscript. Study supported by grants from the National Institutes of Health (GM25653 to J.W.S. and HL-15835 to the Pennsylvania Muscle Institute).
M.
P.
was supported by an Anatomy Training Grant (GM-00281). LITERATURE CITED dos Remedios, C. G., and D. Gilmour 1978 Is there a third type of filament in striated muscles? J. Biochem., 84: 235-238. Fischman, D. A. 1972 Development o f striated muscle. In: The Structure and Function of Muscle, Vol. 1, G. H. Boune, ed., Academic Press, New York, 2nd Ed., pp. 75-148. 268
Goll, D. E . , M. H . Stromer, R. M. Robson, B. M. Luke and K. S. Hammond 1977 E x t r a c t i o n , p u r i f i c a t i o n and l o c a l i z a t i o n of a - a c t i n i n from asynchromous i n s e c t f l i g h t muscle. I n : I n s e c t F l i g h t Muscle, R . T . Tregear, ed. North Holland Publ. Co., Amsterdam, p p . 15-40. Granger, B . L . , and E . Lazarides 1978 The e x i s t e n c e o f an i n s o l u b l e Z-disc s c a f f o l d i n chicken s k e l e t a l muscle. Cell , 15: 1253-1268. Hanson, J . , and H . E . Huxley 1953 S t r u c t u r a l b a s i s of t h e c r o s s s t r i a t i o n s in muscle. Nature, 1 7 2 : 530-532. Hanson, J . , and H . E. Huxley 1955 The s t r u c t u r a l b a s i s o f c o n t r a c t i o n i n s t r i a t e d muscle. Sym. SOC. Exper. Biology, 9: 228-264. Hasselbach, W., and G . Schneider 1951 Der L-Myosin-und Aktingehalt des Kaninchenmuskels. Biochem. Z . , 321: 461-475. Huxley, H . E. 1963 Electron microscopic studies on the s t r u c t u r e of natural and s y n t h e t i c f i l a m e n t s from s t r i a t e d muscle. J . Mol. B i o l . , 7: 281-307. Huxley, H . E. 1968 I n : Symposium on Muscle. Symp. b i o l . H u n g . , 8: 249-250. Huxley, H . E . , and Hanson, J . 1954 Changes i n the c r o s s - s t r i a t i o n of muscle d u r i n g c o n t r a c t i o n and s t r e t c h and t h e i r s t r u c t u r a l i n t e r p r e t a t i o n . Nature, 173: 973-976. Ishikawa, H . , R . Bischoff and H . Holtzer 1968 Mitosis and intermediate. s i z e d f i l a m e n t s in developing muscle. 3 . C e l l . B i o l . , 38: 538-555. Kelly, D . E . 1969 Myofibrillogenesis and Z-band d i f f e r e n t i a t i o n . Anat. Rec., 163: 403-426. Rash, J . E . , J . J . Biesele and G. 0. Gey 1970 Three c l a s s e s of f i l a ments i n c a r d i a c d i f f e r e n t i a t i o n . J . U l t r a s t r u c t . Res., 33: 408-435. Sanger, J . W., J . M. Sanger and J . G w i n n 1978 Analysis o f r o l e s o f a c t i n , microtubules and intermediate f i l a m e n t s i n spreading f i b r o b l a s t s and muscle c e l l s . J . Cell B i o l . , 79: 272a. Somlyo, A . P . , A. V. Somlyo, F. T . Ashton and J . V a l l i e r e s 1976 Vertebrate and smooth muscle: ul t r a s t r u c t u r e and f u n c t i o n . Cold Spring Harbor Conf. Cell P r o l i f . , 3: 165-183. Szent-Gyorgyi, A. G . 1953 Meromyosins, t h e subunits o f myosin. Arch Biochem Biophys., 42: 305-320. Weihing, R . R. 1979 The cytoskeleton and plasma membrane. Meth. Archive. exp. P a t h o l . , 8: 42-109.
269
