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Cell ultrastructure responses to mechanical injury
J.
ULTRASTRUCTURERESEARCH:4, 473-481 (1960)
473
Cell U l t r o s t r u c t u r e Responses to Mechonicol Injury A Preliminory Report z HILTON H. MOLLENHAUER,W. GORDON WHALEY and JAMES H. LEECH
The Plant Research Institute, the University of Texas, Austin, Texas Received August 22, 1960 Responses of cellular organelles to purposely-induced mechanical injury, at or near the surfaces produced by excision of sections from maize root tips, have been studied. These responses are apparent in sections fixed within a few seconds of excision, hence they are essentially immediate. The pores in the nuclear envelope are enlarged and, frequently, proliferation of the nuclear envelope is apparent. The nuclear ground substance appears more granular; the nucleolus appears unchanged. Structural modifications are seen in the mitochondria, endoplasmic reticulum, and the plasma membrane, but there are no striking changes in the Golgi apparatus or the ground substance of the cytoplasm. Certain types of associations, not ordinarily seen in uninjured cells, between some cellular organelles, particularly mitochondria with mitochondria, mitochondria with proplastids, and both of these organelles with "lipid" bodies, are also injury responses. Our studies of the ultrastructure of meristematic cells of the maize root apex have occasionally revealed cells with certain conspicuous modifications of some components. Our attention was first called to these by what seemed to be proliferations of the nuclear envelope (24). Further investigation was suggested by the possibility that this appearance of the nuclear envelope might relate to appearances in other experimental materials in which this structure has been reported to form "blebs," or possibly to give rise to mitochondria or other cytoplasmic organelles (1-15, 17, 18). Investigations have demonstrated that what appears to be proliferation of the nuclear envelope is one of several definite responses to mechanical injury of the cell. Several features of these injury responses are apparent in cells fixed within a few seconds after purposely induced mechanical injury. The progress of the ultrastructural changes induced by such injury will be presented in a later paper. 1 The work reported in this paper has been supported in part by National Institutes of Health Grant RG-7289. It has also utilized equipment provided under ONR Contract Nonr-1438(00).
474
H I L T O NH. MOLLENHAUER, W. GORDON WHALEY AND JAMES H. LEECH MATERIAL AND METHODS
The material was handled in preparation, fixation in potassium permanganate (2% aqueous solution), and electron microscopy as indicated in earlier papers (16, 21, 22). The cells studied were those of layers closest to the surface produced by excision. The injury involved is that caused by the excision. Blocks of material were excised from within 1000/~ of the apex of the root proper. In each instance the material was placed in the fixative within 15 seconds of excision. Thus, the effects recorded are essentially those immediately following injury. Attention also has been directed to the time factor in these injury responses and to possible interrelations among the responses; the data will be presented in another paper.
RESULTS AND DISCUSSION
In this report the injury responses are described simply in terms of their appearance in fixed sections. Fig. 1 is a micrograph of an uninjured cell to the structures of which those of cells showing responses to injury may be compared. Fig. 2 is a micrograph of a cell showing several of the injury responses. Figs. 3-6 illustrate responses in varying degrees of expression. THE NUCLEARENVELOPE
Apparently, certain distinct changes in the nuclear envelope are essentially immediate responses to mechanical injury. One notable change is an increase in the size of the nuclear pores (Fig. 2). This modification gives the nuclear envelope, as seen in section, a decidedly perforate appearance. The nuclear pores in injured cells, except for the enlargement, do not seem to differ from those in normal cells of similar material prepared in the same manner (23, 24). It is possible that additional pores are formed; however, enlargement of existing pores would seem to account for the aspect of the sections studied. Concomitantly, there may be a proliferation of one or more regions of the nuclear envelope outward from the nucleus into the cytoplasm (Fig. 3). These proliferations have a compartmentalizing effect; they do not appear to involve the pore structures
All figures are micrographs of material from the maize root apex fixed in a 2 % aqueous solution of potassium permanganate for two hours at 22°C. The key to the labeling is as follows: er, endoplasmic reticulum; ga, Golgi apparatus; l, lipid body; m, mitochondrion; n, nucleus; pp, proplastid; w, cell wall. FIG. 1. Portions of uninjured meristematic cells showing in section normal appearance of the nuclear envelope, nuclear substance, cytoplasmic ground substance, endoplasmic reticulum, Golgi structures, mitochondria, proplastids, "lipid bodies," and the surfaces of the protoplasts.
Facing page 474.
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of the original nuclear envelope which may often be seen distinctly within the proliferating region. Whether these apparent proliferations of the nuclear envelope involve the envelope alone or segments of endoplasmic reticulum continuous with or adjacent to it, or both, has not been established. Such proliferation may extend for considerable distances into the cytoplasm, even to the surface of the protoplast, and may involve what, seemingly, are segments of endoplasmic reticulum. It has been postulated that the nuclear envelope and the endoplasmic reticulum are components of the same cellular system (19,20, 23, 24). No structural differentiation has been seen in the substance included within the proliferations. The proliferating regions of the nuclear envelope could easily be interpreted as representing the formation by the envelope of mitochondria or other organelles. Manifestations which are apparently comparable have been so interpreted by several investigators (2, 4, 5, 13). The study on which this paper is based has not, however, provided any evidence to support such a conclusion. In some regions the two membranes of the injured nuclear envelope are separated by varying distances much greater than those usually encountered in uninjured cells or in not so modified regions of injured cells (compare Figs. 1 and 2). In such regions the substance between the two membranes occasionally fixes and stains in a manner different from the ground substance of the cytoplasm or the nuclear material. In some instances formed bodies are seen between the two membranes (Fig. 2). Sometimes such bodies appear to be the result of foldings of one or another of the membranes. THE NUCLEUS
The modifications of the nuclear envelope are accompanied by changes in the nuclear material. The nuclear material, apart from the nucleolus, becomes more granular in general appearance and distinct, relatively dark granules become conspicuous in the nuclear ground substance (Fig. 2). There are occasional changes in the shape of the nucleus in injured cells but their relation to normal changes in nuclear shape is not known. Changes in the nucleolus have not been observed. THE CYTOPLASMICGROUNDSUBSTANCE The early changes in nuclear substance are not paralleled by any conspicuous changes in the cytoplasmic ground substance. However, some time after injury, an interval greater than that with which this paper is concerned, the ground substance of the cytoplasm becomes more dense but retains a uniform appearance. THE ENDOPLASMIC RETICULUM
The response to injury of the endoplasmic reticulum is striking, though variable in both character and extent. The system often undergoes changes in form, frequently
476
HILTON H. MOLLENHAUER, W. GORDON WHALEY AND JAMES H. LEECH
compartmentalizing the cytoplasmic ground substance (Fig. 4). Occasionally, the system in section suggests a section through a mass of bubbles. What appear to be responses of the endoplasmic reticulum may parallel, or in some manner may be associated with, responses of the nuclear envelope as well as certain other cytoplasmic organelles. The associations and the reactivity of the endoplasmic reticulum cannot at this time be evaluated as there is not sufficient information concerning the various stages of activity in normal cells. It would be possible to suppose that the endoplasmic reticulum is producing organelles but, as is the case with the nuclear envelope, proof of this has not been produced by this study. THE GOLGI APPARATUS
Some modifications of the Golgi apparatus are apparent immediately after injury. When followed for a longer period, these modifications include a decrease in the larger lamellar components, i.e., the cisternae, of the Golgi apparatus which is accompanied by a marked increase in the number of small vesicles seen (Fig, 2). The Golgi cisternae often disappear completely. OTHER ORaANELI~ES Injury causes some organelles to develop types of associations, not ordinarily apparent in uninjured cells, with other organelles--mitochondria with mitochondria, mitochondria with proplastids, and both with "lipid bodies" (23) (Fig. 5). These organelle associations occasionally involve, variously, the endoplasmic reticulum, the Golgi apparatus, and the plasma membrane. Also, the mitochondria and proplastids tend to be appressed to the protoplast surface, often at those points which may be the loci of intercellular contacts or plasmodesmata. In both the organelle to organelle associations and the mitochondrion and proplastid to protoplast surface associations (Fig. 6), there are distinct changes in form; these changes seem to reflect increased intimacy of association of the organelles. They are apparent particularly
FI~. 2. A portion, in section, of an injured cell of a type comparable to that in Fig. 1. The nuclear pores are enlarged, and the nuclear envelope is otherwise modified (see text). The nuclear substance is generally more granular and shows, in addition, conspicuous, discrete, dense granules. Several of the cytoplasmic organclles show modifications of form, and the surface of the protoplast is modified in some regions. Fla. 3. Proliferation of the nuclear envelope, a, proliferation, in this section at least, confined to the nuclear envelope, b, a proliferation extending to the surface of the protoplast and apparently involving the endoplasmic reticulum. FIG.4. Injury responses of the endoplasmic reticulum, a, showing relationships to the nuclear envelope and the surface of the protoplast, b, showing general distribution in the cytoplasm. Fio. 5. An injury-response association of certain cytoplasmic organelles (see text). FIG. 6 a and b. Injury responses at the surface of the protoplast.
i
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HILTON H. MOLLENHAUER, W. GORDON WHALEY AND JAMES H. LEECH
among the mitochondria which in general assume forms not seen in uninjured cells (compare Figs. 1 and 2, 5, 6). Changes are conspicuous, too, in the "lipid bodies" but these vary greatly in the same general manner in uninjured cells. The form of the modifications of the proplastid appear to be less extensive. In time, there are some distinct changes in the spacings of the bounding membranes of the mitochondria, and the proplastids appear to lose some of their characteristic internal membrane structure. THE PROTOPLAST SURFACE
The plasma membrane is sometimes considerably modified immediately after injury. It is sometimes characterized by what appear to be numerous invaginations and/ or by associations with the endoplasmic reticulum. The resulting complexity does not permit study of it as a distinct membrane (Fig. 6). Sometimes what seem to be discrete formed bodies, or delimited regions of cytoplasm, are seen at this stage outside the protoplast, between it and the cell wall (Fig. 6a). As has been suggested, various organelles may be associated intimately with changes at the surface of the protoplast. The possibility has been noted (23) that the surface of the protoplast of the meristematic cell is involved in activities other than the simple passage of ions and molecules in solution. Further study indicates the protoplast surface to be a highly reactive region. The responses made to injury initially, at least, are localized in as yet undetermined relationships, probably positional. At times they are not confined to the exact site of injury. CONCLUSION The present evidence does not suffice to distinguish the effects described as degenerative effects from responses of the cellular components in an adjustment to injury; nor is it known whether the changes noted enable the cell to maintain some degree of normal activity for a period, or partially to reconstitute itself. Some cells showing these modifications undergo complete degeneration, as will be noted in a later paper. Also, the mechanism by which the type of mechanical injury used here induces these changes is unknown. These preliminary experiments have not yielded evidence of the transmission of injury effects across numbers of cells. In the work so far, the nucleus, the nuclear envelope and the general cellular reticulum make the most striking immediate responses to the mechanical injury; however, profound changes are evident, also, in the Golgi apparatus and some other organelles, and, at times, in the surface of the protoplast.
ULTRASTRUCTURE RESPONSES TO MECHANICALINJURY
48l
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
21. 22. 23. 24.
AFZELIUS,B. A., Z. Zellforsch. u. Mikrosk. Anat. 45, 660 (1957). BARER, R., JOSEPH, S. and MEEK, G. A., Proc. Roy. Soc. London 152, 353 (1960). BARTON,A. A. and CAUSEY, G., J. Anat. 92, 399 (1958). BRANDT, P. W. and PAPPAS, G. D., J. Biophys. Biochem. Cytol. 6, 91 (1959). CAUSEY,G. and HOFFMAN, H., Brit. J. Cancer 9, 666 (1955). COHEN, A. I., J. Biophys. Biochem. Cytol. 3, 859 (1957). GAY, H., Anat. Record 120, 728 (1954). - - - - Proc. Natl. Acad. Sci. U.S. 41, 370 (1955). -Anat. Record 122, 469 (1955). - - - - Cold Spring Harbor Symp. Quant. Biol. 21, 257 (1956). - - - - J. Biophys. Biochem. Cytol. 2, Suppl. 407 (1956). HAGUENAU,F., Intern. Rev. Cytol. 7, 425 (1958). KAUFMANN,B. P. and GAy, H., Nucleus 1, 57 (1958). KAUFMANN, ]3. P., McDoNALD, M. R., GAY, H., GHOSH, C., SILBERZAHN, A. and FUSCALDO, K., Carneige Inst. Wash Yr. Book 54, 255 (1955). KAUFMANN,I . P., GAY, H., DE, O. N. and YOSHIDA,Y.. Carneige Inst. Wash. Yr. Book 56, 378 (1957). MOLLENHAUER,H., J. Biophys. Biochem. Cytol. 6, 431 (1959). REBHON, L. I., J. Biophys. Biochem. Cytol. 2, 93 (1956). SWIFT, H., J. Biophys. Biochem. Cytol. 2, Suppl. 415 (1956). WATSON, M. L., J. Biophys. Biochem. Cytol. 1, 257 (1955). -ibid. 6, 147 (1959). WHALEY,W. G., KEPHART,J. E. and MOLLENHAUER,H. H., Am. J. Botany 46, 743 (1959). --J. Biophys. Biochem. Cytol. 5, 501 (1959). WHALEY,W. G., MOLLENHAUER,H. H., LEECH, J. H., Am. J. Botany 47, 401 (1960). -J. Biophys. Biochem. Cytol. 8, 233 (1960).
ULTRASTRUCTURERESEARCH:4, 473-481 (1960)
473
Cell U l t r o s t r u c t u r e Responses to Mechonicol Injury A Preliminory Report z HILTON H. MOLLENHAUER,W. GORDON WHALEY and JAMES H. LEECH
The Plant Research Institute, the University of Texas, Austin, Texas Received August 22, 1960 Responses of cellular organelles to purposely-induced mechanical injury, at or near the surfaces produced by excision of sections from maize root tips, have been studied. These responses are apparent in sections fixed within a few seconds of excision, hence they are essentially immediate. The pores in the nuclear envelope are enlarged and, frequently, proliferation of the nuclear envelope is apparent. The nuclear ground substance appears more granular; the nucleolus appears unchanged. Structural modifications are seen in the mitochondria, endoplasmic reticulum, and the plasma membrane, but there are no striking changes in the Golgi apparatus or the ground substance of the cytoplasm. Certain types of associations, not ordinarily seen in uninjured cells, between some cellular organelles, particularly mitochondria with mitochondria, mitochondria with proplastids, and both of these organelles with "lipid" bodies, are also injury responses. Our studies of the ultrastructure of meristematic cells of the maize root apex have occasionally revealed cells with certain conspicuous modifications of some components. Our attention was first called to these by what seemed to be proliferations of the nuclear envelope (24). Further investigation was suggested by the possibility that this appearance of the nuclear envelope might relate to appearances in other experimental materials in which this structure has been reported to form "blebs," or possibly to give rise to mitochondria or other cytoplasmic organelles (1-15, 17, 18). Investigations have demonstrated that what appears to be proliferation of the nuclear envelope is one of several definite responses to mechanical injury of the cell. Several features of these injury responses are apparent in cells fixed within a few seconds after purposely induced mechanical injury. The progress of the ultrastructural changes induced by such injury will be presented in a later paper. 1 The work reported in this paper has been supported in part by National Institutes of Health Grant RG-7289. It has also utilized equipment provided under ONR Contract Nonr-1438(00).
474
H I L T O NH. MOLLENHAUER, W. GORDON WHALEY AND JAMES H. LEECH MATERIAL AND METHODS
The material was handled in preparation, fixation in potassium permanganate (2% aqueous solution), and electron microscopy as indicated in earlier papers (16, 21, 22). The cells studied were those of layers closest to the surface produced by excision. The injury involved is that caused by the excision. Blocks of material were excised from within 1000/~ of the apex of the root proper. In each instance the material was placed in the fixative within 15 seconds of excision. Thus, the effects recorded are essentially those immediately following injury. Attention also has been directed to the time factor in these injury responses and to possible interrelations among the responses; the data will be presented in another paper.
RESULTS AND DISCUSSION
In this report the injury responses are described simply in terms of their appearance in fixed sections. Fig. 1 is a micrograph of an uninjured cell to the structures of which those of cells showing responses to injury may be compared. Fig. 2 is a micrograph of a cell showing several of the injury responses. Figs. 3-6 illustrate responses in varying degrees of expression. THE NUCLEARENVELOPE
Apparently, certain distinct changes in the nuclear envelope are essentially immediate responses to mechanical injury. One notable change is an increase in the size of the nuclear pores (Fig. 2). This modification gives the nuclear envelope, as seen in section, a decidedly perforate appearance. The nuclear pores in injured cells, except for the enlargement, do not seem to differ from those in normal cells of similar material prepared in the same manner (23, 24). It is possible that additional pores are formed; however, enlargement of existing pores would seem to account for the aspect of the sections studied. Concomitantly, there may be a proliferation of one or more regions of the nuclear envelope outward from the nucleus into the cytoplasm (Fig. 3). These proliferations have a compartmentalizing effect; they do not appear to involve the pore structures
All figures are micrographs of material from the maize root apex fixed in a 2 % aqueous solution of potassium permanganate for two hours at 22°C. The key to the labeling is as follows: er, endoplasmic reticulum; ga, Golgi apparatus; l, lipid body; m, mitochondrion; n, nucleus; pp, proplastid; w, cell wall. FIG. 1. Portions of uninjured meristematic cells showing in section normal appearance of the nuclear envelope, nuclear substance, cytoplasmic ground substance, endoplasmic reticulum, Golgi structures, mitochondria, proplastids, "lipid bodies," and the surfaces of the protoplasts.
Facing page 474.
ULTRASTRUCTURE RESPONSES TO MECHANICAL INJURY
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of the original nuclear envelope which may often be seen distinctly within the proliferating region. Whether these apparent proliferations of the nuclear envelope involve the envelope alone or segments of endoplasmic reticulum continuous with or adjacent to it, or both, has not been established. Such proliferation may extend for considerable distances into the cytoplasm, even to the surface of the protoplast, and may involve what, seemingly, are segments of endoplasmic reticulum. It has been postulated that the nuclear envelope and the endoplasmic reticulum are components of the same cellular system (19,20, 23, 24). No structural differentiation has been seen in the substance included within the proliferations. The proliferating regions of the nuclear envelope could easily be interpreted as representing the formation by the envelope of mitochondria or other organelles. Manifestations which are apparently comparable have been so interpreted by several investigators (2, 4, 5, 13). The study on which this paper is based has not, however, provided any evidence to support such a conclusion. In some regions the two membranes of the injured nuclear envelope are separated by varying distances much greater than those usually encountered in uninjured cells or in not so modified regions of injured cells (compare Figs. 1 and 2). In such regions the substance between the two membranes occasionally fixes and stains in a manner different from the ground substance of the cytoplasm or the nuclear material. In some instances formed bodies are seen between the two membranes (Fig. 2). Sometimes such bodies appear to be the result of foldings of one or another of the membranes. THE NUCLEUS
The modifications of the nuclear envelope are accompanied by changes in the nuclear material. The nuclear material, apart from the nucleolus, becomes more granular in general appearance and distinct, relatively dark granules become conspicuous in the nuclear ground substance (Fig. 2). There are occasional changes in the shape of the nucleus in injured cells but their relation to normal changes in nuclear shape is not known. Changes in the nucleolus have not been observed. THE CYTOPLASMICGROUNDSUBSTANCE The early changes in nuclear substance are not paralleled by any conspicuous changes in the cytoplasmic ground substance. However, some time after injury, an interval greater than that with which this paper is concerned, the ground substance of the cytoplasm becomes more dense but retains a uniform appearance. THE ENDOPLASMIC RETICULUM
The response to injury of the endoplasmic reticulum is striking, though variable in both character and extent. The system often undergoes changes in form, frequently
476
HILTON H. MOLLENHAUER, W. GORDON WHALEY AND JAMES H. LEECH
compartmentalizing the cytoplasmic ground substance (Fig. 4). Occasionally, the system in section suggests a section through a mass of bubbles. What appear to be responses of the endoplasmic reticulum may parallel, or in some manner may be associated with, responses of the nuclear envelope as well as certain other cytoplasmic organelles. The associations and the reactivity of the endoplasmic reticulum cannot at this time be evaluated as there is not sufficient information concerning the various stages of activity in normal cells. It would be possible to suppose that the endoplasmic reticulum is producing organelles but, as is the case with the nuclear envelope, proof of this has not been produced by this study. THE GOLGI APPARATUS
Some modifications of the Golgi apparatus are apparent immediately after injury. When followed for a longer period, these modifications include a decrease in the larger lamellar components, i.e., the cisternae, of the Golgi apparatus which is accompanied by a marked increase in the number of small vesicles seen (Fig, 2). The Golgi cisternae often disappear completely. OTHER ORaANELI~ES Injury causes some organelles to develop types of associations, not ordinarily apparent in uninjured cells, with other organelles--mitochondria with mitochondria, mitochondria with proplastids, and both with "lipid bodies" (23) (Fig. 5). These organelle associations occasionally involve, variously, the endoplasmic reticulum, the Golgi apparatus, and the plasma membrane. Also, the mitochondria and proplastids tend to be appressed to the protoplast surface, often at those points which may be the loci of intercellular contacts or plasmodesmata. In both the organelle to organelle associations and the mitochondrion and proplastid to protoplast surface associations (Fig. 6), there are distinct changes in form; these changes seem to reflect increased intimacy of association of the organelles. They are apparent particularly
FI~. 2. A portion, in section, of an injured cell of a type comparable to that in Fig. 1. The nuclear pores are enlarged, and the nuclear envelope is otherwise modified (see text). The nuclear substance is generally more granular and shows, in addition, conspicuous, discrete, dense granules. Several of the cytoplasmic organclles show modifications of form, and the surface of the protoplast is modified in some regions. Fla. 3. Proliferation of the nuclear envelope, a, proliferation, in this section at least, confined to the nuclear envelope, b, a proliferation extending to the surface of the protoplast and apparently involving the endoplasmic reticulum. FIG.4. Injury responses of the endoplasmic reticulum, a, showing relationships to the nuclear envelope and the surface of the protoplast, b, showing general distribution in the cytoplasm. Fio. 5. An injury-response association of certain cytoplasmic organelles (see text). FIG. 6 a and b. Injury responses at the surface of the protoplast.
i
480
HILTON H. MOLLENHAUER, W. GORDON WHALEY AND JAMES H. LEECH
among the mitochondria which in general assume forms not seen in uninjured cells (compare Figs. 1 and 2, 5, 6). Changes are conspicuous, too, in the "lipid bodies" but these vary greatly in the same general manner in uninjured cells. The form of the modifications of the proplastid appear to be less extensive. In time, there are some distinct changes in the spacings of the bounding membranes of the mitochondria, and the proplastids appear to lose some of their characteristic internal membrane structure. THE PROTOPLAST SURFACE
The plasma membrane is sometimes considerably modified immediately after injury. It is sometimes characterized by what appear to be numerous invaginations and/ or by associations with the endoplasmic reticulum. The resulting complexity does not permit study of it as a distinct membrane (Fig. 6). Sometimes what seem to be discrete formed bodies, or delimited regions of cytoplasm, are seen at this stage outside the protoplast, between it and the cell wall (Fig. 6a). As has been suggested, various organelles may be associated intimately with changes at the surface of the protoplast. The possibility has been noted (23) that the surface of the protoplast of the meristematic cell is involved in activities other than the simple passage of ions and molecules in solution. Further study indicates the protoplast surface to be a highly reactive region. The responses made to injury initially, at least, are localized in as yet undetermined relationships, probably positional. At times they are not confined to the exact site of injury. CONCLUSION The present evidence does not suffice to distinguish the effects described as degenerative effects from responses of the cellular components in an adjustment to injury; nor is it known whether the changes noted enable the cell to maintain some degree of normal activity for a period, or partially to reconstitute itself. Some cells showing these modifications undergo complete degeneration, as will be noted in a later paper. Also, the mechanism by which the type of mechanical injury used here induces these changes is unknown. These preliminary experiments have not yielded evidence of the transmission of injury effects across numbers of cells. In the work so far, the nucleus, the nuclear envelope and the general cellular reticulum make the most striking immediate responses to the mechanical injury; however, profound changes are evident, also, in the Golgi apparatus and some other organelles, and, at times, in the surface of the protoplast.
ULTRASTRUCTURE RESPONSES TO MECHANICALINJURY
48l
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
21. 22. 23. 24.
AFZELIUS,B. A., Z. Zellforsch. u. Mikrosk. Anat. 45, 660 (1957). BARER, R., JOSEPH, S. and MEEK, G. A., Proc. Roy. Soc. London 152, 353 (1960). BARTON,A. A. and CAUSEY, G., J. Anat. 92, 399 (1958). BRANDT, P. W. and PAPPAS, G. D., J. Biophys. Biochem. Cytol. 6, 91 (1959). CAUSEY,G. and HOFFMAN, H., Brit. J. Cancer 9, 666 (1955). COHEN, A. I., J. Biophys. Biochem. Cytol. 3, 859 (1957). GAY, H., Anat. Record 120, 728 (1954). - - - - Proc. Natl. Acad. Sci. U.S. 41, 370 (1955). -Anat. Record 122, 469 (1955). - - - - Cold Spring Harbor Symp. Quant. Biol. 21, 257 (1956). - - - - J. Biophys. Biochem. Cytol. 2, Suppl. 407 (1956). HAGUENAU,F., Intern. Rev. Cytol. 7, 425 (1958). KAUFMANN,B. P. and GAy, H., Nucleus 1, 57 (1958). KAUFMANN, ]3. P., McDoNALD, M. R., GAY, H., GHOSH, C., SILBERZAHN, A. and FUSCALDO, K., Carneige Inst. Wash Yr. Book 54, 255 (1955). KAUFMANN,I . P., GAY, H., DE, O. N. and YOSHIDA,Y.. Carneige Inst. Wash. Yr. Book 56, 378 (1957). MOLLENHAUER,H., J. Biophys. Biochem. Cytol. 6, 431 (1959). REBHON, L. I., J. Biophys. Biochem. Cytol. 2, 93 (1956). SWIFT, H., J. Biophys. Biochem. Cytol. 2, Suppl. 415 (1956). WATSON, M. L., J. Biophys. Biochem. Cytol. 1, 257 (1955). -ibid. 6, 147 (1959). WHALEY,W. G., KEPHART,J. E. and MOLLENHAUER,H. H., Am. J. Botany 46, 743 (1959). --J. Biophys. Biochem. Cytol. 5, 501 (1959). WHALEY,W. G., MOLLENHAUER,H. H., LEECH, J. H., Am. J. Botany 47, 401 (1960). -J. Biophys. Biochem. Cytol. 8, 233 (1960).