DEVELOPMENTAL
BIOLOGY
48,
487-492
(1976)
Sarcomere
Reorganization JOHN
Department
of Cytology,
F.
in Mite Muscle’
ARONSON’
Dartmouth
Medical
Accepted
September
School,
Hanover,
New
Hampshire
26, 1975
The number of sarcomeres in a given muscle of the mite Tarsonemus randsi was constant in both larval and adult stages, with the exception of the two medial dorsal metapodosomal muscles in males. These muscles have three sarcomeres in larvae and one sarcomere in adults. This change in sarcomere number within a muscle was observed in the living animal by polarized light microscopy using parthenogenetically derived male larvae. Initially the transforming muscles shortened slowly (hours) and the appearance of the sarcomeres was comparable to that seen during normal contraction. With continued shortening there was apposition of adjacent A bands and disappearance of clearly visible Z lines, but no loss of birefringence. Over the next 12 hr there was further shortening of the muscle and loss of birefringence. This was apparent as shortening of the three apposed A regions to the length of a single A band with a small increase in muscle width and no increase in the peak retardation of the birefringent region. The observations are discussed in terms of differential loss of the A filaments of the two terminal sarcomeres.
amorphosis to the adult male. At this time one of these muscles undergoes a biologically determined transformation from a long muscle with three sarcomers to a short muscle with one sarcomere. This event was studied by polarized light microscopy in the living animal.
INTRODUCTION
The striated muscle sarcomere is a characteristically patterned unit of molecularly complex structure whose size and constitution may vary depending on genetic, developmental, and physiological considerations. The number of sarcomeres in a muscle varies both in series and, if we stretch the definition, in parallel. A change in the number of sarcomeres in series is particularly interesting because it reflects a precise local developmental change whose nature can be predicted on the basis of our present knowledge of sarcomere structure. In an earlier study (Aronson, 19611, I described muscles in a tarsomemid mite that had three short sarcomeres. These muscles increased in length by a factor of five during development in a process that presumably involved linear growth of A and of I filaments. It did not involve an increase in the number of sarcomeres. In this report I would like to describe an interesting subsequent change seen during met-
MATERIALS
RESULTS
The initial observation that the medial muscles of the dorsal metapodosoma had 487
c 1976 by Academic Press, Inc. of reproduction in any form reserved
METHODS
The animal studied, Tarsonemus randsi, was raised in the laboratory. Male larvae appear in normal cultures only infrequently and for this reason we used parthenogentically derived males obtained as progeny from single isolated larva. Larvae for study were selected after they had moved off the food onto the side of the culture tube and were quiescent. They were mounted in light mineral oil between a slide and coverslip for observation and were slightly flattened. Observations were made with 546-nm polarized light, changes in birefringence were detected by rotating a thin sheet of mica having about onetenth wavelength maximum retardation and linear measurements were made with a 16X filar micrometer.
’ This work was supported by USPHS Grant No. 10171, by USNSF Grant No. 31739 to S. Inoue and USPHS Grant No. HL-08805 to A. P. Fishman. 2 Present address: Cardiovascular-Pulmonary Division, Hospital of the University of Pennsylvania, 3600 Spruce Street, Philadelphia PA 19104. Copyright All rights
AND
488
DEVELOPMENTAL
BIOLOGY
one sarcomere in adult males and three sarcomeres in young larvae was seen to result from developmental changes occurring in quiescent stage mites. Some of the developmental stages of this animal are shown in Fig. 1, and the appearance of the muscles studied is shown in Fig. 2. Two adult animals with single sarcomere muscles having different A to I band ratios are shown in Figs. 2b and 2c to demonstrate that the muscle behaves functionally as a single sarcomere. Direct observation of actively moving animals showed this most clearly. Polarized light microscopy was used to study the details of this transformation. The same animal could be followed for over 24 hr after mounting in mineral oil, if unflattened and not exposed to excessive light. At the end of this time the mite often broke out of the old exoskeleton and walked from the microscope field. The first changes apparent were a decrease in muscle and sarcomere length without a change
FIG. 1. Developmental (not shown). (b) Adult (upper). x 240.
VOLUME
48, 1976
in A band length, but with some increase in width of the birefringent region (muscle width). This appeared analogous to normal muscle shortening. It occurred at a time when there was lateral separation of the anterior muscle insertions at the intersegmental line and a shift in position or a loss of the posterior insertions. Z lines were readily apparent as narrow isotropic bands in the center of the weakly birefringent I region (Fig. 2a, arrow). With continued shortening the A bands became apposed, the Z line contrast was lost and discrete sarcomeres were not clearly distinguishable by polarized light microscopy. In the 12 hr following the loss of readily visible sarcomere structure there was a further increase in muscle width of about 50%. This was accompanied by a decrease in the length of the muscle and of the birefringent region to a length comparable to that of a single sarcomere and of a single A band. Detailed observations of the birefringent region during this period gave
stages of Tarsonemus ran&i. (a) Egg, which gives rise to the single larval female mite. (c) Postlarval quiescent mite (lower) being carried by an adult
stage male
489
BRIEF NOTES
FIG. 2a. Dorsal metapodosomal muscles in a living larval mite. The two medial muscles are the ones which change sarcomere number. Arrow points to a Z line. Polarized light. x 1000. FIGS. Zb,c. Appearance of dorsal metapodosomal muscles in adult males showing the single sarcomere of a medial muscle for comparison with the three sarcomere medial muscles in Fig. 2a. Polarized light. * 1000.
no evidence of interdigitation of the birefringent A regions which would be apparent as a more birefringent band at the level of each Z line. In one instance differentiation within the birefringent region appeared to show short terminal A bands and a central A band of normal length. Figure 3 shows that the optical retardation measured along the center of the muscle, where it is greatest, did not increase markedly during the time when the length of the birefringent region was decreasing. Also the increase in muscle width during this time was moderate and much less than threefold. Birefringence is shown in Fig. 3 as the change in compensator range used. Although not included in Fig. 3, measurements of retardation of the A region in an adjacent nontransforming sarcomere during this same period showed little or no change with time. The A region in adjacent untransformed muscles did not change length appreciably during the quiescent stage, but an increase in muscle width was apparent (Fig. 3). Such an increase in width was a property of many muscles in both male and female mites during the quiescent period,
6-
MUSCI~E
IJ ,y ~
L-z. TIME
2c Ihours)
oq 3” TIME
,’ ihoilrs)
,(
FIG. 3. The data shown are from two animals. Both left and right transforming muscles in each animal were followed and are shown separately as ( x 1 or (01. Measurements of retardation (shown as compensator angle), width and length ofthe birefringent region (or A regions before they abut) are included. The time of appearance of contractility is shown by the arrows. Data for adjacent nontransforming muscles (0) and (+ 1 are shown by the dashed line (-1 and for transforming muscles c x 1 and (0) by the solid lines. The compensator angle is the difference between field extinction and maximum darkness on the muscle. The broken line (- - -) shows the length of the central A region in adjacent nontransforming muscles
but its extent was less and the time of onset delayed relative to the transforming muscles. During development there was a shift of the dorsal metapodosomal muscles in the
490
DEVELOPMENTAL
BIOLOGY
side and the appearance of overlying refractile material. This caused considerable image deterioration unless the area observed was closely masked. A photographic sequence in a single individual is shown in Fig. 4. In Fig. 4a it is possible to see that there has been lateral movement of the muscles as compared to a larva (Fig. 2a). The rest of the sequence is at a slightly higher magnification and illustrates the process described earlier. Observations of active adult male mites showed that the transformed muscle was functionally a single sarcomere with a constant A band length at a variety of sarcomere lengths. An H gap was apparent at long sarcomere lengths. General evidence that these mite sarcomeres have an intercligitating filament structure is shown in Fig. 5a, where the A and I filaments in a highly stretched muscle appear to have been pulled free of each other, and in Fig. 5b, where a variety of sarcomere lengths are present in the same muscle. The change in sarcomere number seen in male mites did not occur in the female mites nor were other muscles in this animal observed to change sarcomere number. In addition, the number of sarcomeres within a given muscle was strongly fixed. Polarized light observation of muscles in several hundred larvae of indeterminate sex showed no instances of differences in sarcomere number within a given muscle. DISCUSSION
Birefringence is a classic property of the A band (A for anisotropic). On the basis of extraction studies summarized by Hanson and Huxley (1955) we can consider that A band birefringence reflects the organization of myosin in parallel filaments. The amount of birefringent material is directly related to the optical retardation summed over the area. This quantitative property, coupled with the ability to observe changes in birefringence without fixation in a single individual and to associate these changes with known properties of striated
VOLUME
48, 1976
muscle sarcomeres, provides a strong basis for discussing the nature of the reorganization seen. We can initially consider two types of situation: (a) one in which there is complete breakdown of all or most filaments followed by reorganization to a single new sarcomere; or (b) one in which there is differential breakdown of filaments with selective retention of some, either randomly or in terms of sarcomere origin. In this work there is no indication of near total reorganization of the myofibrils, or at least of the strongly birefringent elements, of the sort described for intersegmental muscles in Rhodnius (Wigglesworth, 1956; Toselli and Pepe, 1968). Possible evidence might have been extreme supercontraction and complete loss of birefringence, a decrease in optical retardation, or a more disorganized pattern of birefringence. The observations support a mechanism in which A filaments are lost since there is a decrease in total birefringence (area x retardation). This A filament loss is probably selective and involves A filaments in the terminal sarcomeres only. Evidence for this view is: (a) the lack of an initial increase in retarclation in the central A region followed by a decrease which might have been expected if both lateral sets of A filaments had interdigit&d with those of the central sarcomere; (b) the absence of an overlapping birefringent band pattern of the sort seen during supercontraction (Aronson, 1963); and (c) a single direct observation which appeared to show a shortened terminal A region with a central A region of normal length. There was no visual evidence in the single sarcomere muscle of the two Z lines which were originally present. By implication we consider that the two Z lines and their attached actin filaments have been lost from the myofibril and that the A regions from the central sarcomere are now intercligitating with I filaments which insert in the cell membrane. Evidence that
BRIEF
FIG. 4. Sequence of photographs showing Polarized light with varying compensation.
491
NOTES
the change to a single sarcomere a, x 800; others x 1400.
muscle
in a single
individual.
492
DEVELOPMENTAL
BIOWGY
VOLUME
48,
1976
provide a basis for differential I filament stability. Since there can be considerable plasticity in the size of arthropod muscle sarcomeres within an animal or even within a muscle, it is curious that a muscle has shortened by a decrease in sarcomere number rather than by forming short sarcomeres. REFERENCES
FIG. 5. Larval muscles at different sarcomere lengths. (a) Three highly stretched two sarcomere muscles in which birefringent I filaments appear to have been pulled free of the A region. (b) Partial contraction of a medial metapodosomal muscle which demonstrates dimensional changes associated with sarcomere shortening.
I filaments are those originally present at the membrane insertion is lacking, but it is interesting that recent work on cultured cells (Goldman et al., 1974) suggests that cell-cell contacts affect actin-like filament assemblv at the membrane. This might
ARONSON, J. F. (1961). Sarcomere size in developing muscles of a Tarsonemid mite. J. Biophys. Biothem. Cytol 11, 147-156. ARONSON, J. F. (1963). Overlap of the birefringent component of adjacent A regions during the induced shortening of Drosophila muscle. J. Cell Biol. 19, 107-114. GOLDMAN, R. D., BUSHNELL, A., SCHLOSS, J., and WANG, E. (1974). Contact mediated assembly of actin-like microfilament bundles. Evidence for two states of actin in non-muscle cells. J. Cell Biol. 63, 112a (abstract). HANSON, J., and HUXLEY, H. E. (1955). The structural basis of contraction in striated muscle.
Symp. Sot. Exptl. Biol. 9, 228-264. TOSELLI, P. A., and PEPE, F. A. (1968). The tine structure of the ventral intersegmental abdominal muscles of the insect Rhodnius prolinus during the moulting cycle. J. Cell Biol. 37,462-481. WIGGLESWORTH, V. B. (1956). Formation and involution of striated muscle Sbers during the growth and moulting cycles of Rho&Gus prolinus (Hemiptera). Quart. J. Microscop. Sci. 97, 465-480.
BIOLOGY
48,
487-492
(1976)
Sarcomere
Reorganization JOHN
Department
of Cytology,
F.
in Mite Muscle’
ARONSON’
Dartmouth
Medical
Accepted
September
School,
Hanover,
New
Hampshire
26, 1975
The number of sarcomeres in a given muscle of the mite Tarsonemus randsi was constant in both larval and adult stages, with the exception of the two medial dorsal metapodosomal muscles in males. These muscles have three sarcomeres in larvae and one sarcomere in adults. This change in sarcomere number within a muscle was observed in the living animal by polarized light microscopy using parthenogenetically derived male larvae. Initially the transforming muscles shortened slowly (hours) and the appearance of the sarcomeres was comparable to that seen during normal contraction. With continued shortening there was apposition of adjacent A bands and disappearance of clearly visible Z lines, but no loss of birefringence. Over the next 12 hr there was further shortening of the muscle and loss of birefringence. This was apparent as shortening of the three apposed A regions to the length of a single A band with a small increase in muscle width and no increase in the peak retardation of the birefringent region. The observations are discussed in terms of differential loss of the A filaments of the two terminal sarcomeres.
amorphosis to the adult male. At this time one of these muscles undergoes a biologically determined transformation from a long muscle with three sarcomers to a short muscle with one sarcomere. This event was studied by polarized light microscopy in the living animal.
INTRODUCTION
The striated muscle sarcomere is a characteristically patterned unit of molecularly complex structure whose size and constitution may vary depending on genetic, developmental, and physiological considerations. The number of sarcomeres in a muscle varies both in series and, if we stretch the definition, in parallel. A change in the number of sarcomeres in series is particularly interesting because it reflects a precise local developmental change whose nature can be predicted on the basis of our present knowledge of sarcomere structure. In an earlier study (Aronson, 19611, I described muscles in a tarsomemid mite that had three short sarcomeres. These muscles increased in length by a factor of five during development in a process that presumably involved linear growth of A and of I filaments. It did not involve an increase in the number of sarcomeres. In this report I would like to describe an interesting subsequent change seen during met-
MATERIALS
RESULTS
The initial observation that the medial muscles of the dorsal metapodosoma had 487
c 1976 by Academic Press, Inc. of reproduction in any form reserved
METHODS
The animal studied, Tarsonemus randsi, was raised in the laboratory. Male larvae appear in normal cultures only infrequently and for this reason we used parthenogentically derived males obtained as progeny from single isolated larva. Larvae for study were selected after they had moved off the food onto the side of the culture tube and were quiescent. They were mounted in light mineral oil between a slide and coverslip for observation and were slightly flattened. Observations were made with 546-nm polarized light, changes in birefringence were detected by rotating a thin sheet of mica having about onetenth wavelength maximum retardation and linear measurements were made with a 16X filar micrometer.
’ This work was supported by USPHS Grant No. 10171, by USNSF Grant No. 31739 to S. Inoue and USPHS Grant No. HL-08805 to A. P. Fishman. 2 Present address: Cardiovascular-Pulmonary Division, Hospital of the University of Pennsylvania, 3600 Spruce Street, Philadelphia PA 19104. Copyright All rights
AND
488
DEVELOPMENTAL
BIOLOGY
one sarcomere in adult males and three sarcomeres in young larvae was seen to result from developmental changes occurring in quiescent stage mites. Some of the developmental stages of this animal are shown in Fig. 1, and the appearance of the muscles studied is shown in Fig. 2. Two adult animals with single sarcomere muscles having different A to I band ratios are shown in Figs. 2b and 2c to demonstrate that the muscle behaves functionally as a single sarcomere. Direct observation of actively moving animals showed this most clearly. Polarized light microscopy was used to study the details of this transformation. The same animal could be followed for over 24 hr after mounting in mineral oil, if unflattened and not exposed to excessive light. At the end of this time the mite often broke out of the old exoskeleton and walked from the microscope field. The first changes apparent were a decrease in muscle and sarcomere length without a change
FIG. 1. Developmental (not shown). (b) Adult (upper). x 240.
VOLUME
48, 1976
in A band length, but with some increase in width of the birefringent region (muscle width). This appeared analogous to normal muscle shortening. It occurred at a time when there was lateral separation of the anterior muscle insertions at the intersegmental line and a shift in position or a loss of the posterior insertions. Z lines were readily apparent as narrow isotropic bands in the center of the weakly birefringent I region (Fig. 2a, arrow). With continued shortening the A bands became apposed, the Z line contrast was lost and discrete sarcomeres were not clearly distinguishable by polarized light microscopy. In the 12 hr following the loss of readily visible sarcomere structure there was a further increase in muscle width of about 50%. This was accompanied by a decrease in the length of the muscle and of the birefringent region to a length comparable to that of a single sarcomere and of a single A band. Detailed observations of the birefringent region during this period gave
stages of Tarsonemus ran&i. (a) Egg, which gives rise to the single larval female mite. (c) Postlarval quiescent mite (lower) being carried by an adult
stage male
489
BRIEF NOTES
FIG. 2a. Dorsal metapodosomal muscles in a living larval mite. The two medial muscles are the ones which change sarcomere number. Arrow points to a Z line. Polarized light. x 1000. FIGS. Zb,c. Appearance of dorsal metapodosomal muscles in adult males showing the single sarcomere of a medial muscle for comparison with the three sarcomere medial muscles in Fig. 2a. Polarized light. * 1000.
no evidence of interdigitation of the birefringent A regions which would be apparent as a more birefringent band at the level of each Z line. In one instance differentiation within the birefringent region appeared to show short terminal A bands and a central A band of normal length. Figure 3 shows that the optical retardation measured along the center of the muscle, where it is greatest, did not increase markedly during the time when the length of the birefringent region was decreasing. Also the increase in muscle width during this time was moderate and much less than threefold. Birefringence is shown in Fig. 3 as the change in compensator range used. Although not included in Fig. 3, measurements of retardation of the A region in an adjacent nontransforming sarcomere during this same period showed little or no change with time. The A region in adjacent untransformed muscles did not change length appreciably during the quiescent stage, but an increase in muscle width was apparent (Fig. 3). Such an increase in width was a property of many muscles in both male and female mites during the quiescent period,
6-
MUSCI~E
IJ ,y ~
L-z. TIME
2c Ihours)
oq 3” TIME
,’ ihoilrs)
,(
FIG. 3. The data shown are from two animals. Both left and right transforming muscles in each animal were followed and are shown separately as ( x 1 or (01. Measurements of retardation (shown as compensator angle), width and length ofthe birefringent region (or A regions before they abut) are included. The time of appearance of contractility is shown by the arrows. Data for adjacent nontransforming muscles (0) and (+ 1 are shown by the dashed line (-1 and for transforming muscles c x 1 and (0) by the solid lines. The compensator angle is the difference between field extinction and maximum darkness on the muscle. The broken line (- - -) shows the length of the central A region in adjacent nontransforming muscles
but its extent was less and the time of onset delayed relative to the transforming muscles. During development there was a shift of the dorsal metapodosomal muscles in the
490
DEVELOPMENTAL
BIOLOGY
side and the appearance of overlying refractile material. This caused considerable image deterioration unless the area observed was closely masked. A photographic sequence in a single individual is shown in Fig. 4. In Fig. 4a it is possible to see that there has been lateral movement of the muscles as compared to a larva (Fig. 2a). The rest of the sequence is at a slightly higher magnification and illustrates the process described earlier. Observations of active adult male mites showed that the transformed muscle was functionally a single sarcomere with a constant A band length at a variety of sarcomere lengths. An H gap was apparent at long sarcomere lengths. General evidence that these mite sarcomeres have an intercligitating filament structure is shown in Fig. 5a, where the A and I filaments in a highly stretched muscle appear to have been pulled free of each other, and in Fig. 5b, where a variety of sarcomere lengths are present in the same muscle. The change in sarcomere number seen in male mites did not occur in the female mites nor were other muscles in this animal observed to change sarcomere number. In addition, the number of sarcomeres within a given muscle was strongly fixed. Polarized light observation of muscles in several hundred larvae of indeterminate sex showed no instances of differences in sarcomere number within a given muscle. DISCUSSION
Birefringence is a classic property of the A band (A for anisotropic). On the basis of extraction studies summarized by Hanson and Huxley (1955) we can consider that A band birefringence reflects the organization of myosin in parallel filaments. The amount of birefringent material is directly related to the optical retardation summed over the area. This quantitative property, coupled with the ability to observe changes in birefringence without fixation in a single individual and to associate these changes with known properties of striated
VOLUME
48, 1976
muscle sarcomeres, provides a strong basis for discussing the nature of the reorganization seen. We can initially consider two types of situation: (a) one in which there is complete breakdown of all or most filaments followed by reorganization to a single new sarcomere; or (b) one in which there is differential breakdown of filaments with selective retention of some, either randomly or in terms of sarcomere origin. In this work there is no indication of near total reorganization of the myofibrils, or at least of the strongly birefringent elements, of the sort described for intersegmental muscles in Rhodnius (Wigglesworth, 1956; Toselli and Pepe, 1968). Possible evidence might have been extreme supercontraction and complete loss of birefringence, a decrease in optical retardation, or a more disorganized pattern of birefringence. The observations support a mechanism in which A filaments are lost since there is a decrease in total birefringence (area x retardation). This A filament loss is probably selective and involves A filaments in the terminal sarcomeres only. Evidence for this view is: (a) the lack of an initial increase in retarclation in the central A region followed by a decrease which might have been expected if both lateral sets of A filaments had interdigit&d with those of the central sarcomere; (b) the absence of an overlapping birefringent band pattern of the sort seen during supercontraction (Aronson, 1963); and (c) a single direct observation which appeared to show a shortened terminal A region with a central A region of normal length. There was no visual evidence in the single sarcomere muscle of the two Z lines which were originally present. By implication we consider that the two Z lines and their attached actin filaments have been lost from the myofibril and that the A regions from the central sarcomere are now intercligitating with I filaments which insert in the cell membrane. Evidence that
BRIEF
FIG. 4. Sequence of photographs showing Polarized light with varying compensation.
491
NOTES
the change to a single sarcomere a, x 800; others x 1400.
muscle
in a single
individual.
492
DEVELOPMENTAL
BIOWGY
VOLUME
48,
1976
provide a basis for differential I filament stability. Since there can be considerable plasticity in the size of arthropod muscle sarcomeres within an animal or even within a muscle, it is curious that a muscle has shortened by a decrease in sarcomere number rather than by forming short sarcomeres. REFERENCES
FIG. 5. Larval muscles at different sarcomere lengths. (a) Three highly stretched two sarcomere muscles in which birefringent I filaments appear to have been pulled free of the A region. (b) Partial contraction of a medial metapodosomal muscle which demonstrates dimensional changes associated with sarcomere shortening.
I filaments are those originally present at the membrane insertion is lacking, but it is interesting that recent work on cultured cells (Goldman et al., 1974) suggests that cell-cell contacts affect actin-like filament assemblv at the membrane. This might
ARONSON, J. F. (1961). Sarcomere size in developing muscles of a Tarsonemid mite. J. Biophys. Biothem. Cytol 11, 147-156. ARONSON, J. F. (1963). Overlap of the birefringent component of adjacent A regions during the induced shortening of Drosophila muscle. J. Cell Biol. 19, 107-114. GOLDMAN, R. D., BUSHNELL, A., SCHLOSS, J., and WANG, E. (1974). Contact mediated assembly of actin-like microfilament bundles. Evidence for two states of actin in non-muscle cells. J. Cell Biol. 63, 112a (abstract). HANSON, J., and HUXLEY, H. E. (1955). The structural basis of contraction in striated muscle.
Symp. Sot. Exptl. Biol. 9, 228-264. TOSELLI, P. A., and PEPE, F. A. (1968). The tine structure of the ventral intersegmental abdominal muscles of the insect Rhodnius prolinus during the moulting cycle. J. Cell Biol. 37,462-481. WIGGLESWORTH, V. B. (1956). Formation and involution of striated muscle Sbers during the growth and moulting cycles of Rho&Gus prolinus (Hemiptera). Quart. J. Microscop. Sci. 97, 465-480.
