- Email: [email protected]
Surface structure of negatively stained membranes
171
Structure of membranes REFERENCES 1. 2. 3. 4.
CHANCE, B., PERRY, R., Acmxnfm, L. and THORELL, B., Reu. Sci. Insfr. 30, 735 (1959). KOHEN, E., Biochim. Biophys. Acfa 75, 139 (1963). ROBBINS, E., J. Gen. Physiof. 43, 853 (1960). THOMISON, J. G., Expff CeU Res. 35, 213 (1964).
SURFACE
STRUCTURE W. P. CUNNINGHAM,
Department
of Biological
OF NEGATIVELY J. W. STILES Sciences, Purdue University,
STAINED
MEMBRANES
and F. L. CRANE Lafayette,
Ind.,
U.S.A.
Received May 6, 1965
N EGATIVE
staining of mitochondria isolated from many cell types reveals numerous spherical particles attached by slender stalks to the surface and edges of the inner membrane [2, 4, 6, 8, IO]. The postulate that these structures contain the electron transport system led to the name, elementary particles. We now find that the other membraneous elements found in cellular homogenates possess distinctive surface features when treated with phosphotungstate stain. SjGstrand al. [7] observed two types of membranes in mitochondrial preparations. One type was described as grey ribbons edged by stalked particles, whereas the other with smaller particles along the edges. No special type was “intensely white tubes” significance was attributed to the two membrane types. \Ve have observed similar membrane types in our large scale preparations of beef heart mitochondria, especially in the light membrane fractions. An electron micrograph of an area of a light mitochondrial fraction showing both types of membrane is presented in Fig. 1. In order to determine whether both of these types of membranes originate from the mitochondria, or whether one type might represent contaminating fragments of some other organelle, we have isolated and purified mitochondria and microsomes from beef heart and from rat and rabbit liver. Tissues were homogenized in 0.5 M sucrose buffered at pH 7.2 with tris HCl. Fractions were separated by differential centrifugation and washed by repeated sedimentation and re-centrifugation. The heavy mitochondria isolated from rabbit liver by this procedure exhibited the normal cytochrome absorption spectrum and electron transport enzyme activities. When these mitochondria were suspended in distilled water and stained with PTA, the edges of all of the membranes were packed with 90 a, stalked particles. None of the “white” tubules could be found. The microsomal fraction, however sedimented from the mitochondrial supernatant and washed three times contained a large number
et
1 This \vork was done during the tenure of a NIH Postdoctoral to W. P. C. and an NIH career investigatorship to F. I,. C. It Teas supported by a grant from the American Heart Association 62G4. Experimenfaf
Ceil Research 40
172
W. P. Cunningham,
J. W. Stiles and F. L. Crane
of these PTA repellent tubules. By far the most predominant component of this fraction were circular or irregular shaped structure 100 to 300 rnp across that appear to be deflated vesicles or flattened membrane fragments. Since these vesicles sometimes appear to be continuous with one or more of the “white” tubules, they may represent different forms of the same membrane type. Some of the microsomal membranes are shown in Fig. 2. The surfaces of the two forms have different appearances after negative staining. The rolled tubes have smooth surfaces which are not penetrated by the PTA and their sharply defined edges are usually surrounded by particles 50-60 A in diameter. Occasionally, they are surrounded by a thin, continuous sheath 20-30 A in width and separated from the vesicle by a PTA filled space 30-50 8, wide. This may mean that the 50-60 A particles are really rounded up fragments of this sheath. Another type of vesicle is observed which does not show projections from the surface. These flattened membranes have rough surfaces which stain with PTA. The tightly packed granules arc about 25-30 A in diameter. They protrude from the surface so that the vesicles have very rough edges. The structures around the edges of both of these type of membranes are easily distinguishable from those seen in mitochondria, being much smaller. Furthermore, neither type of microsomal granule appears in our pictures to be attached to the membrane by a stalk. Microsomes of all of the tissues studied show a similar variety of structure after PTA treatment. None of the striking heterogeneity seen in the negatively stained microsomes is apparent after osmium staining and sectioning. In the latter case, all of the membrane types appear as uniform vesicles ranging in size from 100 to 300 mp. Ribosomes are present in the preparation but are rarely associated with the membranes. Since there are at least three possible sources of the so-called microsomal fraction in the cell, e.g., the rough and smooth endoplasmic reticulum (E.R.) or the plasma membrane (51, it is not surprising that we find several forms of membranes in this material. The absorption spectrum of this fraction shows reduced peaks at 561, 557, 528 and 426 but no peak in the cytochrome (I region at 605. Hased on an A e red-ox b, concentration is 3.8 ~~moles,‘mg protein. We feel, at 557 rnp of 16, the cytochrome therefore, that a major portion of these membranes are derived from the E.R. and that a negligible amount of mitochondrial material is present. Note the mitochondrial membrane stripped of all but two or three of its stalked particles in Fig. 2. It may well be, as Sjiistrand et nl. have suggested, that the particles seen on the surfaces of negatively stained membranes do not represent structures present in uivo. On the other hand, separation of knob-like units from mitochondria by physical treatment 11, $11would imply the selective release of discrete material associated with the membrane surface even if not in knob form in vivo. 1Ve would like to suggest that the various cellular membranes have specific and easily recognizable surface structures after PTA treatment. These characteristic
Fig. I.-Light photungstic 1000 A.
membrane fraction (ETP) from beef heart mitochondria acid. M, mitochondrial membrane; WT, white tubules;
Fig. Z.-Rabbit liver microsomal preparation U’T, white tubule; GM, granular membrane; Experimental
Cell Research 40
stained as above. y 160,000.
stained with 2 ‘I; phosx 160,000 marker equals
A4, mitochondrial
membrane;
173
Structure of membranes
Rsperimenlal
Cell Research
40
174
Bonnie Sue Ebstein et al.
structures, whether artifacts or not, provide one of the quickest and most sensitive methods of identifying isolated membranes in cellular homogenates. They may also be useful in attempts to interpret molecular arrangement of the original cellular membranes. REFERENCES 1. 2. 3. 4. 5. 6. 7.
CHANCE, B., PAKSONS, D. F. and WILLI.~MS, G. R., Science 143, 136 (1964). FERNANDEZ-MORAN, H., Circufafion 26, 1036 (1962). FERNANDEZ-MORAN, H., ODA, T., BLAIR, P. U., and GREEN, D. E., J. Cell Hiol. 22, 63 (1964). NADAKAVUKAREX, M. J., J. Cell Biol. 23, 193 (1964). PALADE, G. and SIEKE~ITZ, P., J. Biophys. Biochem. Cgfol. 2, Ii1 (1956). PARSONS, D. F., Science 140, 985 (1963). SJBSTRAND, F. S., ANDERSON-CEDERGREN, E. and KARLSSON, LT., Suture 202, lOi (1964).
8. SMITH, D. S., J. Cell BioZ. 19, 115 (1963). 9. STASNY, .J. T. and 10. STOECKENIUS, \v.,
CRAM& F. L., .I. Cell Biol. 22, 49 (1964). J. Cell BiOl. 17, 443 (1963).
CELLS FROM ISOLATED
BONNIE
SUE
BLASTOMERES OF 1LYANASSA IN TISSUE CULTURE1
EBSTEIN,z MIRIAM R. L. DEHAAN
D. ROSENTHAL
OBSOLETA
and
Department of Biology, Yale University, New Haven, Corm., Department of Biology, Brandeis University, Waltham, Mass., and Department of Embryology, Carnegie Institution of Washington, Baltimore, Md., Received
May
U.S.A.
14, 1965
1~ 1904 Wilson [7] noted that CD or D blastomeres of Dentalium, isolated in sea water, often tend to adhere to the bottom of the dish. In these cases, he observed a few cells migrating out of the attached embryos onto the glass surface. Clement reported the same thing with Ilyanassa embryos [I]. The present investigation was designed to extend these observations by applying tissue culture techniques and media to isolated blastomeres of Ilyanassa, in order to determine whether molluscan cells can be isolated from the embryo and cultured in vitro. Egg capsules containing embryos at late-trefoil or early 2-cell stage were sterilized by a brief dip (2-3 set) in 70 per cent ethanol. The capsules were opened and the eggs removed to pasteurized sea-water. One blastomere of each egg was punctured with a steel needle, leaving the other intact [l]. 15 to 25 AB or CD blastomeres were transferred to 2 ml of culture medium in 35 mm plastic tissue culture dishes (Falcon 1 Work performed at the Marine Biological Laboratory, Woods Hole, Massachusetts, during the summer of 1964. 2 Supported by a Public Health Service Training Grant in Developmental Biology from the National Institute of Child Health and Human Development; sponsor, Dr J. P. Trinkaus.
Experimental
Cell Research 40
Structure of membranes REFERENCES 1. 2. 3. 4.
CHANCE, B., PERRY, R., Acmxnfm, L. and THORELL, B., Reu. Sci. Insfr. 30, 735 (1959). KOHEN, E., Biochim. Biophys. Acfa 75, 139 (1963). ROBBINS, E., J. Gen. Physiof. 43, 853 (1960). THOMISON, J. G., Expff CeU Res. 35, 213 (1964).
SURFACE
STRUCTURE W. P. CUNNINGHAM,
Department
of Biological
OF NEGATIVELY J. W. STILES Sciences, Purdue University,
STAINED
MEMBRANES
and F. L. CRANE Lafayette,
Ind.,
U.S.A.
Received May 6, 1965
N EGATIVE
staining of mitochondria isolated from many cell types reveals numerous spherical particles attached by slender stalks to the surface and edges of the inner membrane [2, 4, 6, 8, IO]. The postulate that these structures contain the electron transport system led to the name, elementary particles. We now find that the other membraneous elements found in cellular homogenates possess distinctive surface features when treated with phosphotungstate stain. SjGstrand al. [7] observed two types of membranes in mitochondrial preparations. One type was described as grey ribbons edged by stalked particles, whereas the other with smaller particles along the edges. No special type was “intensely white tubes” significance was attributed to the two membrane types. \Ve have observed similar membrane types in our large scale preparations of beef heart mitochondria, especially in the light membrane fractions. An electron micrograph of an area of a light mitochondrial fraction showing both types of membrane is presented in Fig. 1. In order to determine whether both of these types of membranes originate from the mitochondria, or whether one type might represent contaminating fragments of some other organelle, we have isolated and purified mitochondria and microsomes from beef heart and from rat and rabbit liver. Tissues were homogenized in 0.5 M sucrose buffered at pH 7.2 with tris HCl. Fractions were separated by differential centrifugation and washed by repeated sedimentation and re-centrifugation. The heavy mitochondria isolated from rabbit liver by this procedure exhibited the normal cytochrome absorption spectrum and electron transport enzyme activities. When these mitochondria were suspended in distilled water and stained with PTA, the edges of all of the membranes were packed with 90 a, stalked particles. None of the “white” tubules could be found. The microsomal fraction, however sedimented from the mitochondrial supernatant and washed three times contained a large number
et
1 This \vork was done during the tenure of a NIH Postdoctoral to W. P. C. and an NIH career investigatorship to F. I,. C. It Teas supported by a grant from the American Heart Association 62G4. Experimenfaf
Ceil Research 40
172
W. P. Cunningham,
J. W. Stiles and F. L. Crane
of these PTA repellent tubules. By far the most predominant component of this fraction were circular or irregular shaped structure 100 to 300 rnp across that appear to be deflated vesicles or flattened membrane fragments. Since these vesicles sometimes appear to be continuous with one or more of the “white” tubules, they may represent different forms of the same membrane type. Some of the microsomal membranes are shown in Fig. 2. The surfaces of the two forms have different appearances after negative staining. The rolled tubes have smooth surfaces which are not penetrated by the PTA and their sharply defined edges are usually surrounded by particles 50-60 A in diameter. Occasionally, they are surrounded by a thin, continuous sheath 20-30 A in width and separated from the vesicle by a PTA filled space 30-50 8, wide. This may mean that the 50-60 A particles are really rounded up fragments of this sheath. Another type of vesicle is observed which does not show projections from the surface. These flattened membranes have rough surfaces which stain with PTA. The tightly packed granules arc about 25-30 A in diameter. They protrude from the surface so that the vesicles have very rough edges. The structures around the edges of both of these type of membranes are easily distinguishable from those seen in mitochondria, being much smaller. Furthermore, neither type of microsomal granule appears in our pictures to be attached to the membrane by a stalk. Microsomes of all of the tissues studied show a similar variety of structure after PTA treatment. None of the striking heterogeneity seen in the negatively stained microsomes is apparent after osmium staining and sectioning. In the latter case, all of the membrane types appear as uniform vesicles ranging in size from 100 to 300 mp. Ribosomes are present in the preparation but are rarely associated with the membranes. Since there are at least three possible sources of the so-called microsomal fraction in the cell, e.g., the rough and smooth endoplasmic reticulum (E.R.) or the plasma membrane (51, it is not surprising that we find several forms of membranes in this material. The absorption spectrum of this fraction shows reduced peaks at 561, 557, 528 and 426 but no peak in the cytochrome (I region at 605. Hased on an A e red-ox b, concentration is 3.8 ~~moles,‘mg protein. We feel, at 557 rnp of 16, the cytochrome therefore, that a major portion of these membranes are derived from the E.R. and that a negligible amount of mitochondrial material is present. Note the mitochondrial membrane stripped of all but two or three of its stalked particles in Fig. 2. It may well be, as Sjiistrand et nl. have suggested, that the particles seen on the surfaces of negatively stained membranes do not represent structures present in uivo. On the other hand, separation of knob-like units from mitochondria by physical treatment 11, $11would imply the selective release of discrete material associated with the membrane surface even if not in knob form in vivo. 1Ve would like to suggest that the various cellular membranes have specific and easily recognizable surface structures after PTA treatment. These characteristic
Fig. I.-Light photungstic 1000 A.
membrane fraction (ETP) from beef heart mitochondria acid. M, mitochondrial membrane; WT, white tubules;
Fig. Z.-Rabbit liver microsomal preparation U’T, white tubule; GM, granular membrane; Experimental
Cell Research 40
stained as above. y 160,000.
stained with 2 ‘I; phosx 160,000 marker equals
A4, mitochondrial
membrane;
173
Structure of membranes
Rsperimenlal
Cell Research
40
174
Bonnie Sue Ebstein et al.
structures, whether artifacts or not, provide one of the quickest and most sensitive methods of identifying isolated membranes in cellular homogenates. They may also be useful in attempts to interpret molecular arrangement of the original cellular membranes. REFERENCES 1. 2. 3. 4. 5. 6. 7.
CHANCE, B., PAKSONS, D. F. and WILLI.~MS, G. R., Science 143, 136 (1964). FERNANDEZ-MORAN, H., Circufafion 26, 1036 (1962). FERNANDEZ-MORAN, H., ODA, T., BLAIR, P. U., and GREEN, D. E., J. Cell Hiol. 22, 63 (1964). NADAKAVUKAREX, M. J., J. Cell Biol. 23, 193 (1964). PALADE, G. and SIEKE~ITZ, P., J. Biophys. Biochem. Cgfol. 2, Ii1 (1956). PARSONS, D. F., Science 140, 985 (1963). SJBSTRAND, F. S., ANDERSON-CEDERGREN, E. and KARLSSON, LT., Suture 202, lOi (1964).
8. SMITH, D. S., J. Cell BioZ. 19, 115 (1963). 9. STASNY, .J. T. and 10. STOECKENIUS, \v.,
CRAM& F. L., .I. Cell Biol. 22, 49 (1964). J. Cell BiOl. 17, 443 (1963).
CELLS FROM ISOLATED
BONNIE
SUE
BLASTOMERES OF 1LYANASSA IN TISSUE CULTURE1
EBSTEIN,z MIRIAM R. L. DEHAAN
D. ROSENTHAL
OBSOLETA
and
Department of Biology, Yale University, New Haven, Corm., Department of Biology, Brandeis University, Waltham, Mass., and Department of Embryology, Carnegie Institution of Washington, Baltimore, Md., Received
May
U.S.A.
14, 1965
1~ 1904 Wilson [7] noted that CD or D blastomeres of Dentalium, isolated in sea water, often tend to adhere to the bottom of the dish. In these cases, he observed a few cells migrating out of the attached embryos onto the glass surface. Clement reported the same thing with Ilyanassa embryos [I]. The present investigation was designed to extend these observations by applying tissue culture techniques and media to isolated blastomeres of Ilyanassa, in order to determine whether molluscan cells can be isolated from the embryo and cultured in vitro. Egg capsules containing embryos at late-trefoil or early 2-cell stage were sterilized by a brief dip (2-3 set) in 70 per cent ethanol. The capsules were opened and the eggs removed to pasteurized sea-water. One blastomere of each egg was punctured with a steel needle, leaving the other intact [l]. 15 to 25 AB or CD blastomeres were transferred to 2 ml of culture medium in 35 mm plastic tissue culture dishes (Falcon 1 Work performed at the Marine Biological Laboratory, Woods Hole, Massachusetts, during the summer of 1964. 2 Supported by a Public Health Service Training Grant in Developmental Biology from the National Institute of Child Health and Human Development; sponsor, Dr J. P. Trinkaus.
Experimental
Cell Research 40