- Email: [email protected]
Formation of whirling aggregates by Labyrinthula vegetative cells
412
Preliminary notes
liver cells in vivo [7]. This suggested that References the nuclear alterations related to aging may 1. Hay, R J & Strehler, B L, Exp geronto12 (1967) 123. be similar both in vitro and in vivo. In 2. Macieira-Coelho, A, Gerontology 22 (1976) 3. 3. Kaji, K & Matsuo, M, Exp cell res 119 (1979) 231. mouse cells, however, the proportion of 4C 4. Saksela, E & Moorhead, P S, Proc natl acad sci US 50 (1963) 390. nuclei increases sharply until about 2.5 5. Kaji, K & Matsuo, M, Exp gerontol 13 (1978) 439. months after birth, remains unchanged from 6. Yanishevsky, R, Mendelsohn, M L, Mayall, B H & Cristofalo, V J, J cell physiol 84 (1974) 165. 2.5 to 14 months, and then decreases. 7. Shima, A & Sugahara, T, Exp gerontol 11 (1976) As shown by the polyploidization index, 193. 8. Kaji, K & Matsuo, M, Mech ageing develop 8 chick cells are considerably polyploidized (1978) 233. in the last stage of culture and the sharp May 22, 1980 increase in polyploidization at the end of Received Revised version received September 30, 1980 their lifespan may suggest that the forma- Accepted October 9, 1980 tion of polyploid cells is a result and not a cause of aging. It should be noted that in chick cells, the proportion of 4C nuclei Copyright 0 IYXI by Academic Pres. Inc. All rights of reproduction m any form reserved increases with advancing PD level, al00 14-4827/RI10204I2-06%02.Kl/0 though population doubling rate remains constant until the 12th PD level (fig. 2). Formation of whirling aggregates The increase may reflect the progress of by Labyrinthda vegetative cells NOR10 NAKATSUJI,’ TAKAKO NAKATSUJI an age-related nuclear alteration. and EUGENE BELL,* Department of Biology, MasIn our previous report [3], we discussed sachusetts Institute of Technology, Cambridge, MA the possibility that, i.e., a polyploid nucleus 02139, and Marine Biological Laboratory, Woods may result from failure of cytokinesis fol- Hole, MA 02543, USA lowed by nuclear fusion or that DNA rep- Summary. A novel form of aggregation of the vegecells of the colonial marine protist Labyrinthala lication may occur in a cell in which both tative has been analysed cinematically and induced reprocytokinesis and karyokinesis have failed. ducibly in vitro by addition of gelatin to the growth Vegetative cells are observed to converge The data from the time-course experiments medium. spirally to a center and continue to move circularly of age-dependent polyploidization are com- around the center with a mean angular velocity of lSO”/min, suggesting the existence of a gradient of patible with both the above hypotheses. chemoattractant. The possibility is discussed that the However, we have observed previously system of actin rails located in the slimeways in which move is arranged circularly to permit the obthat about 3 % of the chick cells were bi- cells served movements. nuclear at the early stage of culture, increasing to 20% at the end of their lifespan The marine slime mold Labyrinthula forms [8]. Since the percentage of 4C nuclei is colonies in which spindle-shaped vegetative larger in chick cells at each PD level than cells are enveloped by slimeways organized the percentage of binuclear cells, it is un- into a network [4, 6, 7, 93. The material likely that a 4C nucleus is produced in a of the slimeways is secreted by the spindle binuclear cell bearing two 2C nuclei. Fur- cells and contains actin and myosin as ther studies using a variety of methods are major components but no organelles such necessary to elucidate the mechanism of as mitochondria, Golgi, or ribosomes [4]. age-dependent polyploidization. We thank Professor V. J. Cristofalo and MS M. Hoffman of the Wistar Institute for their kind and help ful advice on the preparation of the manuscript. Exp Cell Res 131 (1981)
’ Present address: Deoartment of Anatomv. George Washington University Medical Center, W&hingto& DC 20037, USA. * To whom offprint requests may be addressed. Prmted
in Sweden
Preliminary
We have shown that actin is organized into a system of rails in the slimeways and proposed a model to explain the unique motility of vegetative cells which have no visible means of locomotion [4]. The model suggests that actin filaments of the slimeway interact with actin filaments associated with each spindle cell to produce a gliding motion. The slimeways are also motile and responsible for colony expansion. They exhibit both filopodial and lamellipodial activity. The contractility of the slimeways as well as the locomotion of the spindle cells appear to be controlled by Ca2+ [3]: glycerinated slimeways contract when Ca2+ and ATP are added. With ionophore A23 187 contraction is induced at a Ca2+ concentration of 1x lop7 M Ca2+. In studying the life cycle of Labyrinthala, which appears to consist of three stages [5]: (1) vegetative growth in the course of which cell number and colony size increase; (2) aggregation of cells into sot-i; and (3) zoospore differentiation, we encountered a novel mode of cell aggregation previously noticed but not analysed [lo, 111. The phenomenon is characterized by cell whirling and is easily seen through the microscope at a magnification of x50. The formation of whirling aggregates at various loci within colonies suggests, as in the cellular slime molds, the development of gradients of an attractant substance. In this paper we report on the laboratory conditions required to initiate the whirling aggregation and detail some of its features. Materials
and Methods
Isolation and maintenance of Labyrinrhula spp. have been described [4,8]. To stimulate formation of whirling aggregates, colonies were grown in dishes containing sea water with 1% gelatin, 1% horse serum, 0.4% glucose, 0.2% sodium glutamate, 2 mgfl thiamine and antibiotics. Gelatin was essential for inducing aggregation. Colonies on the.dish surface expand and, after 4 or 5 days, several centers of whirling
notes
413
aggregates were observed per 6 cm dish. Aggregates were observed in 75% of the dishes containing the medium described above. Dishes with no gelatin contained no aggregates. The aggregates persist for about 1 week, then transform into stable aggregates which can form sori and in which cells are quiescent. For scanning electron microscopy @EM), colonies were fixed with 2% glutaraldehyde in 0.2 M sodium cacodylate buffer, pH 6.8 for 30 rnin at room temperature, rinsed in the same buffer and post-lixed with 1% 0~0, in the same buffer for 15 min at room temperature. They were critical-point-dried through liquid carbon dioxide. The methods of glycerination and decoration with myosin S-l fragment and thin sectioning [4], as well as the method for time-lapse cinematography [3] have been described.
Results and Discussion
The whirling movement of spindle cells is easily seen at a magnification of x50 or X100. Sequential frames from time-lapse films show clearly that the spindle cells move in spiral or circular pathways (fig. 1). The center of the aggregating cell mass is tightly packed with cells and the entire mass rotates with a mean angular velocity of about 150”lmin. Spindle cells located near but outside the rotating mass converge
Fig. I. Serial photographs printed from a 16 mm film, showing a center of whirling aggregation. The central mass of spindle cells rotates clockwise. Its longer axis is shown by a bar. Spindle cells near the central mass also rotate, including a small cell aggregate (X) and a single cell (arrow), while cells far from the center, such as a cell mass (O), move slowly. Frames minted are senarated by intervals of 20 sec. Bar, 5Opm. _ Fig. 2. SEMs showing whirling aggregates. (a) A central cell mass making a mound. Cells are covered by a continuous membrane. (b) The margin of a region of aggregation whose center is to the right, outside the photograph, is shown by arrows. The region of aggregation itself appears to consist of a sheet of slimeway material, to the left of which are normal slimeways (arrowheads). (c) A center as it appears from the undersurface. Spindle cells remain attached to the dorsal membrane which curled during fixation (asterisks). (d) A higher magnification view of the cells attached to the undersurface. Note fibers that connect spindle cells. Bar, 5 pm. Fig. 4. A thin section of slimeways that was glycerinated and decorated with myosin S-l fragments. Six nm fuaments inside slimeways are decorated in arrowhead configuration. An intersection of several slimeways have an expanded area where the filaments curve. Bar, 0.1 pm.
414
Preliminary notes
Preliminary notes
415
4 16
Preliminary
notes
Fig. 3. A diagram showing a whirling aggregate. The central cell mass is rotating clockwise. Spindle cells approach it spirally after streaming into the region of aggregation through thick slimeways. The aggregation area is an expanded slimeway.
possible. Normally colonies grow as discs which expand radially by radial outgrowth of slimeways. But within the disc, radially oriented slimeways are interconnected by numerous criss-crossing slimeway tracks, providing many small circuits. Furthermore, new slimeway outgrowths can and do arise from any existing slimeway element not only at the colony periphery but anywhere within the colony as well. Hence the normal arrangement of filaments at slimeway intersections (fig. 4) might easily be converted to a circular track. While centripetal cell movement, i.e. the movement of cells which lie outside the aggregate into an aggregate, suggests diffusion of an attractant from the aggregate center along slimeway trails no evidence has yet been presented for a gradient of attractant, although chemotactic behavior of vegetative spindle cells is seen in relation to food such as yeast, toward which colonies stream actively. Both chemotaxis, of course, and circular or spiral movements of cells are known to occur during the aggregation phase of the cellular slime mold
spirally or centripetally into it (figs 1, 3). Adjacent whirling centers sometimes fuse to make a larger aggregate which continues to increase in size up to 1 mm as spindle cells from outside the whirling aggregate stream into it. Scanning electron microscopy (SEM) shows that the center cells pile up into a mound (fig. 2a) and that the entire whirl- r1,21. ing aggregate is covered by a membrane, The authors thank Dennis Solomon for technical aspossibly of the same nature as that which sistance. Supported by Grant no. NOAA Sea Grant invests the slimeway (fig. 2b). During fixa- 04-7-158-44079. tion the dorsal slimeway membrane frequently breaks and curls up, thus exposing the undersurface to which the spindle cells References 1. Clark, R L & Steck, T L, Science 204 (1979) 1163. remain attached (fig. 2~). Fibers which con2. Durston, A J & Vork, F, J cell sci 36 (1979) 261. nect these cells (fig. 2d) may be actin fila3. Nakatsuji, N &Bell, E, Cell motility 1 (1980). 17. 4. Nakatsuji, N, Sher, S, Solomon, D, Nakatsuji, T ments which are shown to be the major & Bell, E, J cell biol. In press. component of slimeways [4]. 5. Olive, L S, The mycetozoans, pp, 215-241. Academic Press, New York (1975). These photographs suggest that the entire Perkins, F 0, Arch microbial 84 (1972) 95. aggregation occurs in one expanded region 4. Porter, D, Protoplasma 67 (1%9) 1. 8: Solomon, D, Sher, S & Bell, E, Oceans’ 78, p. 99. of slimeway in which many spindle cells Fourth annual combined conference of the Marine move circularly as shown in fig. 3. It is Technology Society and the Institute of Electrical and Electronics Engineers (1978). not clear what causes the spiral or circular 9. Stey, H, Z Zellforsch 102 (1969) 419. movement. We can propose that actin fila- 10. Watson, S W, Ph.D. Thesis, Univ Wisconsin (1957). ments are arranged circularly to make it Exr, Cell Res 131 (1981)
Preliminary 11. Pokomy, K S, Ph.D. Thesis, Columbia Univ (1971). Received June 6, 1980 Revised version received September 24, 1980 Accepted September 29, 1980
Copyright @ 1981 hy Academic Press. Inc. All rights of reproduction in any form reserved 0014.4827/8l/OZO417-03$02.00/O
The maternal inheritance of proteins synthesized in mammalian mitochondria RANDALL W. YATSCOFF,’ JEREMY R. MASON,’ LARRY W. BELBECKZ and KARL B. FREEMAN,’ ‘Department of Biochemistry and $Department of Pathology, McMaster ton, Ont L.8N 325, Canada
University,
Hamil-
Interspecific variations between the proteins synthesized in the mitochondria of two closely related-mammalian species were demonstrated to be matemallv inherited bv examination of their hybrid progeny. -This, in accbrdance with the documented &parental inheritance of mitochondrial DNA (mtDNA) would indicate that the latter codes for the products of mitochondrial translation.
Summary.
The mitochondrial genome of lower eukaryotes has been shown to code for a limited number of RNA and protein species [ 1, 21. These cytoplasmic genes have been identified by reciprocal crosses in which they are transmitted only from the female parent [3]. In contrast, in higher eukaryotes, although the mitochondrial genome has been found to code for poly A-containing RNA species, it has not been directly ascertained whether this RNA is translated into mitochondrial protein [4] and uniparental inheritance of mitochondrial DNA (mtDNA) has only been demonstrated by means of molecular techniques [5-91. For example, Hutchison et al. [6] utilized difference between the restriction fragment patterns of the mtDNAs of the horse, Equus caballus, and the donkey, E. asinus, to show that reciprocal hybrids (mules and hinnies) had no (less than 5 %) paternal mtDNA. Printed
in Sweden
notes
417
Previously we and others have shown that between 10 and 13 mitochondrial translation products are synthesized by whole mammalian cells [l&13]. Interspecific variation in the number and electrophoretic mobilities of these proteins has been demonstrated, which could reflect known difference in the mtDNA from species to species [4, 14, 151.Alternatively, if the mRNA is coded in the nucleus but transported into and translated within mitochondria, this variation could reflect the divergence of the nuclear genome amongst species. Utilizing the known maternal inheritance of mtDNA and electrophoretic difference in the proteins synthesized in mitochondria of zebra (E. zebra) and donkey (E. asinus), the genes coding for these proteins were shown to be in mtDNA by characterization of the mitochondrial proteins synthesized by their hybrid, the zeedonk. Materials
and Methods
Biopsy samples were taken from the neck of a female donkey, from a male zebra and from their hybrid, a male zeedonk. The samples were explanted in Petri dishes and then crown as adherent cultures at 37°C in a-minimal es&tial medium (MEM) [16] supplemented with 15% (v/v) fetal calf serum (FCS). Approx. 3 months after explantation, lo8 cells from each suecies were harvested and labelled with PSLmethionine (1000 Ci mmol-I) as previously de&ibid [lo]. This was carried out in the presence of either 300 pg cycloheximide ml-l to specifically inhibit protein synthesis on cytosolic ribosomes or cycloheximide plus 150 pg Tevenel ml-’ to additionally inhibit mitochondrial translation. The mitochondria were isolated from these labelled cells [lo] and their polypeptides analysed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis incorporating 12.5% acrylamide monomer and utilizing the buffer system of Laemmli [17]. Fluorography was performed on the resulting gels [18].
Results
Fig. la shows a fluorogram of mitochondrial proteins from donkey cells labelled in the presence of cycloheximide, to permit mitochondrial translation only. Thirteen components were identified as products of Exp CellRrs 131 (1981)
Preliminary notes
liver cells in vivo [7]. This suggested that References the nuclear alterations related to aging may 1. Hay, R J & Strehler, B L, Exp geronto12 (1967) 123. be similar both in vitro and in vivo. In 2. Macieira-Coelho, A, Gerontology 22 (1976) 3. 3. Kaji, K & Matsuo, M, Exp cell res 119 (1979) 231. mouse cells, however, the proportion of 4C 4. Saksela, E & Moorhead, P S, Proc natl acad sci US 50 (1963) 390. nuclei increases sharply until about 2.5 5. Kaji, K & Matsuo, M, Exp gerontol 13 (1978) 439. months after birth, remains unchanged from 6. Yanishevsky, R, Mendelsohn, M L, Mayall, B H & Cristofalo, V J, J cell physiol 84 (1974) 165. 2.5 to 14 months, and then decreases. 7. Shima, A & Sugahara, T, Exp gerontol 11 (1976) As shown by the polyploidization index, 193. 8. Kaji, K & Matsuo, M, Mech ageing develop 8 chick cells are considerably polyploidized (1978) 233. in the last stage of culture and the sharp May 22, 1980 increase in polyploidization at the end of Received Revised version received September 30, 1980 their lifespan may suggest that the forma- Accepted October 9, 1980 tion of polyploid cells is a result and not a cause of aging. It should be noted that in chick cells, the proportion of 4C nuclei Copyright 0 IYXI by Academic Pres. Inc. All rights of reproduction m any form reserved increases with advancing PD level, al00 14-4827/RI10204I2-06%02.Kl/0 though population doubling rate remains constant until the 12th PD level (fig. 2). Formation of whirling aggregates The increase may reflect the progress of by Labyrinthda vegetative cells NOR10 NAKATSUJI,’ TAKAKO NAKATSUJI an age-related nuclear alteration. and EUGENE BELL,* Department of Biology, MasIn our previous report [3], we discussed sachusetts Institute of Technology, Cambridge, MA the possibility that, i.e., a polyploid nucleus 02139, and Marine Biological Laboratory, Woods may result from failure of cytokinesis fol- Hole, MA 02543, USA lowed by nuclear fusion or that DNA rep- Summary. A novel form of aggregation of the vegecells of the colonial marine protist Labyrinthala lication may occur in a cell in which both tative has been analysed cinematically and induced reprocytokinesis and karyokinesis have failed. ducibly in vitro by addition of gelatin to the growth Vegetative cells are observed to converge The data from the time-course experiments medium. spirally to a center and continue to move circularly of age-dependent polyploidization are com- around the center with a mean angular velocity of lSO”/min, suggesting the existence of a gradient of patible with both the above hypotheses. chemoattractant. The possibility is discussed that the However, we have observed previously system of actin rails located in the slimeways in which move is arranged circularly to permit the obthat about 3 % of the chick cells were bi- cells served movements. nuclear at the early stage of culture, increasing to 20% at the end of their lifespan The marine slime mold Labyrinthula forms [8]. Since the percentage of 4C nuclei is colonies in which spindle-shaped vegetative larger in chick cells at each PD level than cells are enveloped by slimeways organized the percentage of binuclear cells, it is un- into a network [4, 6, 7, 93. The material likely that a 4C nucleus is produced in a of the slimeways is secreted by the spindle binuclear cell bearing two 2C nuclei. Fur- cells and contains actin and myosin as ther studies using a variety of methods are major components but no organelles such necessary to elucidate the mechanism of as mitochondria, Golgi, or ribosomes [4]. age-dependent polyploidization. We thank Professor V. J. Cristofalo and MS M. Hoffman of the Wistar Institute for their kind and help ful advice on the preparation of the manuscript. Exp Cell Res 131 (1981)
’ Present address: Deoartment of Anatomv. George Washington University Medical Center, W&hingto& DC 20037, USA. * To whom offprint requests may be addressed. Prmted
in Sweden
Preliminary
We have shown that actin is organized into a system of rails in the slimeways and proposed a model to explain the unique motility of vegetative cells which have no visible means of locomotion [4]. The model suggests that actin filaments of the slimeway interact with actin filaments associated with each spindle cell to produce a gliding motion. The slimeways are also motile and responsible for colony expansion. They exhibit both filopodial and lamellipodial activity. The contractility of the slimeways as well as the locomotion of the spindle cells appear to be controlled by Ca2+ [3]: glycerinated slimeways contract when Ca2+ and ATP are added. With ionophore A23 187 contraction is induced at a Ca2+ concentration of 1x lop7 M Ca2+. In studying the life cycle of Labyrinthala, which appears to consist of three stages [5]: (1) vegetative growth in the course of which cell number and colony size increase; (2) aggregation of cells into sot-i; and (3) zoospore differentiation, we encountered a novel mode of cell aggregation previously noticed but not analysed [lo, 111. The phenomenon is characterized by cell whirling and is easily seen through the microscope at a magnification of x50. The formation of whirling aggregates at various loci within colonies suggests, as in the cellular slime molds, the development of gradients of an attractant substance. In this paper we report on the laboratory conditions required to initiate the whirling aggregation and detail some of its features. Materials
and Methods
Isolation and maintenance of Labyrinrhula spp. have been described [4,8]. To stimulate formation of whirling aggregates, colonies were grown in dishes containing sea water with 1% gelatin, 1% horse serum, 0.4% glucose, 0.2% sodium glutamate, 2 mgfl thiamine and antibiotics. Gelatin was essential for inducing aggregation. Colonies on the.dish surface expand and, after 4 or 5 days, several centers of whirling
notes
413
aggregates were observed per 6 cm dish. Aggregates were observed in 75% of the dishes containing the medium described above. Dishes with no gelatin contained no aggregates. The aggregates persist for about 1 week, then transform into stable aggregates which can form sori and in which cells are quiescent. For scanning electron microscopy @EM), colonies were fixed with 2% glutaraldehyde in 0.2 M sodium cacodylate buffer, pH 6.8 for 30 rnin at room temperature, rinsed in the same buffer and post-lixed with 1% 0~0, in the same buffer for 15 min at room temperature. They were critical-point-dried through liquid carbon dioxide. The methods of glycerination and decoration with myosin S-l fragment and thin sectioning [4], as well as the method for time-lapse cinematography [3] have been described.
Results and Discussion
The whirling movement of spindle cells is easily seen at a magnification of x50 or X100. Sequential frames from time-lapse films show clearly that the spindle cells move in spiral or circular pathways (fig. 1). The center of the aggregating cell mass is tightly packed with cells and the entire mass rotates with a mean angular velocity of about 150”lmin. Spindle cells located near but outside the rotating mass converge
Fig. I. Serial photographs printed from a 16 mm film, showing a center of whirling aggregation. The central mass of spindle cells rotates clockwise. Its longer axis is shown by a bar. Spindle cells near the central mass also rotate, including a small cell aggregate (X) and a single cell (arrow), while cells far from the center, such as a cell mass (O), move slowly. Frames minted are senarated by intervals of 20 sec. Bar, 5Opm. _ Fig. 2. SEMs showing whirling aggregates. (a) A central cell mass making a mound. Cells are covered by a continuous membrane. (b) The margin of a region of aggregation whose center is to the right, outside the photograph, is shown by arrows. The region of aggregation itself appears to consist of a sheet of slimeway material, to the left of which are normal slimeways (arrowheads). (c) A center as it appears from the undersurface. Spindle cells remain attached to the dorsal membrane which curled during fixation (asterisks). (d) A higher magnification view of the cells attached to the undersurface. Note fibers that connect spindle cells. Bar, 5 pm. Fig. 4. A thin section of slimeways that was glycerinated and decorated with myosin S-l fragments. Six nm fuaments inside slimeways are decorated in arrowhead configuration. An intersection of several slimeways have an expanded area where the filaments curve. Bar, 0.1 pm.
414
Preliminary notes
Preliminary notes
415
4 16
Preliminary
notes
Fig. 3. A diagram showing a whirling aggregate. The central cell mass is rotating clockwise. Spindle cells approach it spirally after streaming into the region of aggregation through thick slimeways. The aggregation area is an expanded slimeway.
possible. Normally colonies grow as discs which expand radially by radial outgrowth of slimeways. But within the disc, radially oriented slimeways are interconnected by numerous criss-crossing slimeway tracks, providing many small circuits. Furthermore, new slimeway outgrowths can and do arise from any existing slimeway element not only at the colony periphery but anywhere within the colony as well. Hence the normal arrangement of filaments at slimeway intersections (fig. 4) might easily be converted to a circular track. While centripetal cell movement, i.e. the movement of cells which lie outside the aggregate into an aggregate, suggests diffusion of an attractant from the aggregate center along slimeway trails no evidence has yet been presented for a gradient of attractant, although chemotactic behavior of vegetative spindle cells is seen in relation to food such as yeast, toward which colonies stream actively. Both chemotaxis, of course, and circular or spiral movements of cells are known to occur during the aggregation phase of the cellular slime mold
spirally or centripetally into it (figs 1, 3). Adjacent whirling centers sometimes fuse to make a larger aggregate which continues to increase in size up to 1 mm as spindle cells from outside the whirling aggregate stream into it. Scanning electron microscopy (SEM) shows that the center cells pile up into a mound (fig. 2a) and that the entire whirl- r1,21. ing aggregate is covered by a membrane, The authors thank Dennis Solomon for technical aspossibly of the same nature as that which sistance. Supported by Grant no. NOAA Sea Grant invests the slimeway (fig. 2b). During fixa- 04-7-158-44079. tion the dorsal slimeway membrane frequently breaks and curls up, thus exposing the undersurface to which the spindle cells References 1. Clark, R L & Steck, T L, Science 204 (1979) 1163. remain attached (fig. 2~). Fibers which con2. Durston, A J & Vork, F, J cell sci 36 (1979) 261. nect these cells (fig. 2d) may be actin fila3. Nakatsuji, N &Bell, E, Cell motility 1 (1980). 17. 4. Nakatsuji, N, Sher, S, Solomon, D, Nakatsuji, T ments which are shown to be the major & Bell, E, J cell biol. In press. component of slimeways [4]. 5. Olive, L S, The mycetozoans, pp, 215-241. Academic Press, New York (1975). These photographs suggest that the entire Perkins, F 0, Arch microbial 84 (1972) 95. aggregation occurs in one expanded region 4. Porter, D, Protoplasma 67 (1%9) 1. 8: Solomon, D, Sher, S & Bell, E, Oceans’ 78, p. 99. of slimeway in which many spindle cells Fourth annual combined conference of the Marine move circularly as shown in fig. 3. It is Technology Society and the Institute of Electrical and Electronics Engineers (1978). not clear what causes the spiral or circular 9. Stey, H, Z Zellforsch 102 (1969) 419. movement. We can propose that actin fila- 10. Watson, S W, Ph.D. Thesis, Univ Wisconsin (1957). ments are arranged circularly to make it Exr, Cell Res 131 (1981)
Preliminary 11. Pokomy, K S, Ph.D. Thesis, Columbia Univ (1971). Received June 6, 1980 Revised version received September 24, 1980 Accepted September 29, 1980
Copyright @ 1981 hy Academic Press. Inc. All rights of reproduction in any form reserved 0014.4827/8l/OZO417-03$02.00/O
The maternal inheritance of proteins synthesized in mammalian mitochondria RANDALL W. YATSCOFF,’ JEREMY R. MASON,’ LARRY W. BELBECKZ and KARL B. FREEMAN,’ ‘Department of Biochemistry and $Department of Pathology, McMaster ton, Ont L.8N 325, Canada
University,
Hamil-
Interspecific variations between the proteins synthesized in the mitochondria of two closely related-mammalian species were demonstrated to be matemallv inherited bv examination of their hybrid progeny. -This, in accbrdance with the documented &parental inheritance of mitochondrial DNA (mtDNA) would indicate that the latter codes for the products of mitochondrial translation.
Summary.
The mitochondrial genome of lower eukaryotes has been shown to code for a limited number of RNA and protein species [ 1, 21. These cytoplasmic genes have been identified by reciprocal crosses in which they are transmitted only from the female parent [3]. In contrast, in higher eukaryotes, although the mitochondrial genome has been found to code for poly A-containing RNA species, it has not been directly ascertained whether this RNA is translated into mitochondrial protein [4] and uniparental inheritance of mitochondrial DNA (mtDNA) has only been demonstrated by means of molecular techniques [5-91. For example, Hutchison et al. [6] utilized difference between the restriction fragment patterns of the mtDNAs of the horse, Equus caballus, and the donkey, E. asinus, to show that reciprocal hybrids (mules and hinnies) had no (less than 5 %) paternal mtDNA. Printed
in Sweden
notes
417
Previously we and others have shown that between 10 and 13 mitochondrial translation products are synthesized by whole mammalian cells [l&13]. Interspecific variation in the number and electrophoretic mobilities of these proteins has been demonstrated, which could reflect known difference in the mtDNA from species to species [4, 14, 151.Alternatively, if the mRNA is coded in the nucleus but transported into and translated within mitochondria, this variation could reflect the divergence of the nuclear genome amongst species. Utilizing the known maternal inheritance of mtDNA and electrophoretic difference in the proteins synthesized in mitochondria of zebra (E. zebra) and donkey (E. asinus), the genes coding for these proteins were shown to be in mtDNA by characterization of the mitochondrial proteins synthesized by their hybrid, the zeedonk. Materials
and Methods
Biopsy samples were taken from the neck of a female donkey, from a male zebra and from their hybrid, a male zeedonk. The samples were explanted in Petri dishes and then crown as adherent cultures at 37°C in a-minimal es&tial medium (MEM) [16] supplemented with 15% (v/v) fetal calf serum (FCS). Approx. 3 months after explantation, lo8 cells from each suecies were harvested and labelled with PSLmethionine (1000 Ci mmol-I) as previously de&ibid [lo]. This was carried out in the presence of either 300 pg cycloheximide ml-l to specifically inhibit protein synthesis on cytosolic ribosomes or cycloheximide plus 150 pg Tevenel ml-’ to additionally inhibit mitochondrial translation. The mitochondria were isolated from these labelled cells [lo] and their polypeptides analysed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis incorporating 12.5% acrylamide monomer and utilizing the buffer system of Laemmli [17]. Fluorography was performed on the resulting gels [18].
Results
Fig. la shows a fluorogram of mitochondrial proteins from donkey cells labelled in the presence of cycloheximide, to permit mitochondrial translation only. Thirteen components were identified as products of Exp CellRrs 131 (1981)