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The fine structure of Phycomyces
© 1968 by Academic Press Inc.
s. ULTRASTRUCTURERtSEARCH21, 269--280 (1968)
The Fine Structure of
269
Phycomyces
I. A u t o p h a g i c Vesicles z ROBERT M. THORNTON
Division of Natural Sciences, University of California, Santa Cruz, California 95060 Received August 16, 1967 The fungus Phycomyces blakesleeanus was examined at several developmental stages. The sporangiophores contain vesicles with either a single membrane or a pair of concentric membranes. Such vesicles contain either intact cytoplasm with organelles or a complex form of debris. These findings suggest that certain endoplasmic cisternae of the sporangiophore are engaged in the isolation and lysis of cytoplasm. Longitudinal sections of the sporangiophore reveal axial groupings of vesicles suggesting formation of vesicles near the cell tip, maturation in lower regions, and final discharge of the lytic debris into the large sporangiophore vacuole. The presence of large (above 1 #) granules in vesicles, cytoplasm, and nuclei; the frequent proximity of endoplasmic cisternae to nuclei; the occurrence of nuclei with widely separated inner and outer membranes; and the extension of nuclear membranes into the cytoplasm suggest that the vesicle membranes originate from nuclear membranes. These phenomena were found in all stages of sporangiophore development but not in the vegetative hyphae. Attention was turned in this laboratory a few years ago to the fine structure of the fungus Phycomyces blakesleeanus in hopes of detecting an organelle that could be associated with the sensitive light-growth responses (5) of the sporangiophore. The first result of this work was the detection of "crystal-containing bodies" reminiscent of those in the light-sensitive coleoptiles of Arena (24). A second result has been the finding that the vacuoles of sporangiophores invariably contain debris suggestive of lysed cytoplasm. The origin of this debris is the subject of the present report, and evidence will be presented that the sporangiophore systematically isolates and degrades parts of its own cytoplasm and discards the residues in the vacuole. A similar autophagic system has been reported in the zygomycete GilbertelIa (2, 3), and hydrolytic enzymes are known to occur in vesicular particles in Neurospora (14) 1 This investigation was supported by predoctoral National Science Foundation Fellowships to author and by grant No. GB 3241 from the National Science Foundation to Professor K. V. Thimann. the
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ROBERT M. THORNTON
and in the vacuoles of yeast cells (15). These recent reports on fungal materials are paralleled by growing evidence for lytic structures in plant cells, either as lysosomelike organelles (12, 19, 25) or as autophagic systems related to the formation of vacuoles (13, 20). However, autophagy is far better known in animal cells; the extensive literature has recently been summarized by Gahan (9) and by de Duve and Wattiaux (8). The consensus expressed in both reviews is that autophagy may turn out to be significant in the control of cellular activities, especially during differentiation. That autophagy should occur in the sporangiophore of Phycomyces bIakesleeanus is of special interest in this regard, because the control of growth in this giant cell has been the subject of very precise studies for half a century (see 1, 5, 6, 18). Indeed, it was proposed a decade ago (7), on quantitative grounds, that the bulk of the "growth precursor" that finds its way into the growing region of the sporangiophore is converted into "waste" and is not used directly in growth. The findings reported here may well bear out the latter prediction and should be useful in subsequent work on the role of autophagy in differentiation. These results have been briefly reported elsewhere (22).
MATERIALS AND METHODS
Phycomyces blakesleeanus, strain G5(+) (collected and identified by Dr. H. E. Gruen) was maintained in pure culture on petri dishes of potato-dextrose-agar (21). The cultures were incubated at temperatures of 13, 19, or 24°C either in complete darkness or with continuous light from overhead laboratory fluorescent lamps. Ctdtures were examined and specimens manipulated with the aid of a Bausch and Lomb binocular dissecting microscope at 15 or 25 diameters. For higher resolution, a Zeiss phase microscope was employed. Specimens were fixed for electron microscopy either by excising hyphae and sporangiophores and immersing them in small vials of fixative, or by flooding entire cultures with fixative and delaying excision until after fixation was terminated. Preliminary studies indicated that cells fixed with permanganate were too brittle to carry through the subsequent stages of preparation. The present results were obtained by means of glutaraldehyde fixation, according to the following technique. Specimens were fixed for 5-13 hours at room temperature in 5% glutaraldehyde in 0.1 M phosphate buffer at pH 6-6.3. After several washes in plain buffer, the specimens were postfixed in 1% OsO~ in a similar phosphate buffer for 2-4 hours at room temperature, washed in distilled water, dehydrated with acetone and embedded in Epon or Araldite (16). The use of propylene oxide in embedding was abandoned after preliminary indications of associated damage to the cytoplasm of the sporangiophores. The resin-infiltrated specimens were arranged in flat aluminum foil boats before polymerization. In the cured blocks, both hyphae and sporangiophores could then be selected and oriented in the microtome chuck with the aid of a dissecting microscope allowing the preparation of longitudinal- or cross sections of the cells. The orientation of specimens was
PHYCOMYCES I. AUTOPHAGIC VESICLES
271
further checked by examination of thick (1 tt) sections under the light microscope after staining with toluidine blue. Thin sections were stained with lead citrate and uranyl acetate (16) and examined with an RCA EMU 3H electron microscope.
OBSERVATIONS Each sporangiophore originates as a lateral bulge on a short, swollen terminal hypha that is club-shaped and packed with yellowish, vesicular cytoplasm (10, 21). The outgrowth of the sporangiophore is accompanied by the initiation and continual expansion of a large vacuole that confines the protoplasm of the sporangiophore to a peripheral layer against the cylindrical cell wall. Before sporulation, the apex of the sporangiophore tapers smoothly to a rounded tip. Of the tapering portion, the uppermost (distal to the mycelium) 100-200 # of the sporangiophore is filled with a mass of cytoplasm. Eventually the extreme apex of the cell balloons into a terminal sporangium within which sporulation occurs. Afterward the sporangiophore continues to elongate for many hours (4). The apical 100-200 # of the sporangiophore was found to be rich in vesicles that are variable in shape, bounded by a unit membrane and filled with debris of a composite granular, membranous and globular character. Such vesicles occur both before (Fig. 1) and after (Fig. 2) sporulation. The sporangiophore vacuole was found to contain debris like that in the cytoplasmic vesicles. While this is true before sporulation (Fig. 3), much more marked accumulations of debris in the vacuole were to be seen after sporulation (Fig. 4). In both stages of development, the vacuolar membrane appears to fuse with many cytoplasmic vesicles. In most such instances debris was found in the attached vesicles as well as the adjacent regions of the vacuole (Figs. 3 and 4). Examination of longitudinal sections revealed an orderly arrangement of debriscontaining vesicles and possibly related structures along the axis of the sporangiophore before sporulation. Several zones have been distinguished, as follows. The lowest region, around the head of the vacuole (termed zone D, 100-200 # below the cell tip) is rich in vesicles that average several microns in diameter (Fig. 3). These vesicles contain loosely packed debris, have a single membrane, and are generally either spherical or cusped as if recently derived by the fusion of a pair of spherical vesicles. The acropetally adjacent region (zone C, 50-150 # in length) contains large numbers of amorphous vesicles measuring about 1 or 2 tt in the largest dimension. In each vesicle a single membrane surrounds a tightly packed mass of debris (Fig. 1). Centered about 25 # from the cell tip is a region (zone B) containing several types
272
ROBERT M. THORNTON
of vesicular structure that will be discussed in detail below in connection with the origin of debris-containing vesicles. Finally, the extreme apex (uppermost 10-20 #) of the sporangiophore (zone A) contains few of the vesicles under consideration here. The cytoplasm of the sporangiophore is in general p o o r in the elongated cisternae that are customarily regarded as endoplasmic reticulum in other organisms. The cytoplasm is rich in small cisternae, however. Groups or masses of cisternae, apparently reticular in character, lined with granules of ribosomal dimensions, were c o m m o n l y encountered close to nuclei in sporangiophores before sporulation (Fig. 10). The perinuclear positioning of these cisternae was c o m m o n enough to suggest that, with appropriate sectioning, each of the m a n y nuclei in the sporangiophore would prove to be associated with a mass of endoplasmic cisternae. Reticulae occur remote f r o m nuclei as well, and after sporulation tracts of reticular cisternae spanning m a n y microns were f o u n d (Fig. 2). T h r o u g h o u t the mycelium and sporangiophore, the cytoplasm contains abundant structures composed of an electron dense, fibrous core surrounded by a single membrane (Fig. 5). Such "dense bodies" appear either circular or rod-like, with dimensions of the order of 0.2-0.5/z. Their nature, origin, and function have not yet been determined.
Key to abbreviations
a c d
autophagic (debris-containing) vesicle endoplasmic cisterna debris db dense body dm debris resembling degraded mitochondrion g granular particle h hyphal vesicle
m mitochondrion n nucleus ng nuclear granule np extension of nuclear membrane t microtubule V vacuole of sporangiophore
The bar on each micrograph represents one micron. All magnifications are approximate. Subject material
Figs. 1, 3, 5-11: sporangiophores of length 0.75M mm, before sporulation Fig. 2, 4: sporangiophores after sporulation Fig. 12: growing tip of a hypha Fig. 13: swollen hypha (nongrowing) from which a sporangiophore has arisen Fig. 14: sporangiophore primordium of length 0.1 mm Fro. 1. A debris-containing vesicle in zone C. Fro. 2. Groups of debris-containing vesicles and endoplasmic cisternae in the growing zone after sporulation. FIG. 3. The tip of the sporangiophore and the adjacent cytoplasm of zone D, illustrating the fusion of vesicles with the vacuole and consequent addition of debris to the vacuole. Note the more swollen appearance of vesicles here than in zone C (Fig. 1). FIG. 4. Vacuole and adjacent cytoplasm of the growing zone after sporulation, showing the large quantities of debris that accumulate in the vacuole.
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While dense bodies almost invariably occur free in the cytoplasm, occasionally a dense body and its surrounding cytoplasmic matrix were found enclosed within a membranous sac. The sac consisted in some cases of a pair of concentric unit membranes (Figs. 6, 9, and 10), in other cases a single membrane or a compromise involving areas of single and double membrance structure (Fig. 7). Such figures were found chiefly in zone B, though instances occurred in zone C as well. Less commonly, mitochondria with surrounding cytoplasmic matrix were seen enclosed within a membrane sheath (Fig. 8). Primarily in zone B, areas were often encountered in which endoplasmic cisternae partially surround a portion of cytoplasm (Fig. 7). These partial surrounds, as well as the complete cytoplasm-filled sacs described in the previous paragraph, were invariably found near nuclei, though, since zone B is rich in nuclei (23), this may not be significant. In the intact cytoplasm, in the vacuole, and in the lumens of debris-containing vesicles there occurred electron dense, granular particles averaging several microns in diameter (g in Figs. 3, 9 and 13). Sections of these particles are commonly either circular or kidney-shaped (as in Fig. 13) suggesting that the three-dimensional shape often resembles an indented ball. The origin of these particles became a matter of interest when it was found that nuclei sometimes contain particles of similar granulation and electron density (Fig. 11). Occasionally, nuclei were found in which the separation of the inner and outer membrane was irregular (as in Fig. 11). In some cases a space as wide as a micron intervened between the two membranes of the nuclear sheath. The possibility that these separations were artifacts seems to be excluded by the fact that the majority of nuclei had uniformly separated membranes. In contrast to the sporangiophores, no debris-containing vesicles could be found in the vegetative hyphae. Abundant vesicles are present in the hyphal apices, but these invariably contain nothing more than a variety of electron-dense globular material (Fig. 12). Each hypha vesicle possesses a single limiting membrane, and the hyphae exhibit no sacs enclosing cytoplasm or organelles. The absence of debris-containing vesicles in the vegetative mycelium raises a question concerning the point in differentiation at which such vesicles begin to be made. Examining extremely young sporangiophores (e.g., 100 # long; cf. Figs. 13 and 14), FIo. 5. Several dense bodies and fortuitously related microtubules in zone B. FIG. 6. A vesicle in zone B, composed of two membranes surrounding a mass of ground cytoplasm in which a dense body is embedded. Fro. 7. A region of cytoplasm (a) partially surrounded by endoplasmic cisternae. The enclosed plasm contains a dense body and small vesicles typical of cytoplasm (in contrast to nucleoplasm), and the cisternal membranes are more narrowly spaced than the membranes of nuclei. Zone B. F~. 8. A mitochondrion enclosed within a vesicle in zone C.
276
ROBERT M. THORNTON
the cytoplasm of the swollen parental hypha was found to contain numerous vesicles many of which contain membranous debris (Fig. 13). In these vesicles the debris consists entirely of nested membranes resembling highly distorted mitochondria embedded in fluid space within a surrounding membrane. The primordial sporangiophore apparently owes the first vestige of its vacuole to the fusion of a few such large vesicles containing small amounts of membranous and granular debris (Fig. 14). The electron-dense globular material characteristic of vesicles in the older sporangiophores only begins to appear at a later time, and granular particles of the type that are subsequently prominent in vesicles and vacuole are initially present in the cytoplasm but not in the vesicles of the swollen hypha (Fig. 13). DISCUSSION The evidence bearing on the deposition of debris in the vacuoles of sporangiophores may be divided into several parts for interpretation as follows. (a) Vesicles may be lined with either one or two membranes. In either case, the contents of the vesicle may be either debris or cytoplasm with recognizable dense bodies, large granular particles, or mitochondria. However, in general the vesicles which show only a single membrane are filled with debris, while those with two membranes contain "intact" cytoplasm. Furthermore, regions of cytoplasm are found which are partly surrounded by endoplasmic cisternae. The simplest interpretation for these observations is that certain endoplasmic cisternae come to envelop regions of cytoplasm. If this is true the earliest complete vesicles should possess a double membrane around a mass of intact cytoplasm. Lysis of the contents would follow, together with a reduction from two membranes to one. This proposal matches the mechanism for formation of autophagic vesicles in animal materials, as recently reviewed (8, 9), except that in animal materials the parental cisternae could more clearly be identified as endoplasmic reticulum. It seems appropriate, therefore, to apply the term "autophagic vesicle" to the debris-containing vesicles in the sporangiophore. The validity of this nomenclature is based on the enclosure of organelles within sacs, and no comment can yet be made on the presence of hydrolytic enzymes within the vesicles (cf. 8). However, the presence of hydrolytic enzymes in the vacuoles of higher plant cells (13) suggests a broad uniformity here. FIG. 9. A debris-containing vesicle the double envelope of which clearly consists of a pair of unit membranes. FIG. 10. A nucleus in zone B, showing an extension of the membrane into the cytoplasm. Note also the small group of perinuclear cisternae (c). FI~. 11. A nucleus in zone B, containing a granular particle and having irregularly separated inner and outer membranes. FIG. 12. Cytoplasm about 200/~ behind the apex of a growing hypha of the vegetative mycelium.
°# E
278
R O B E R T M. T H O R N T O N
In addition, the presence of large granular particles in both nuclei and vesicles raises the possibility that some of the debris-containing vesicles might be nuclei in which the observed separation of inner and outer membranes is followed by lysis of the nucleoplasm. This mechanism cannot account for the incorporation of mitochondria and dense bodies into vesicles, however, and does not explain the presence of granular particles in the cytoplasm. (b) Spatially, the sporangiophore apex is organized so that the most apical regions (zone B in particular), contain most of the double-membraned vesicles and instances of partially enveloped cytoplasm. Lower regions contain predominantly single-membraned vesicles filled with debris. Vesicles of the latter type, as judged by the appearance of the contained debris, swell and fuse with the membrane of the large sporangiophore vacuole in zone D (about 100-200 ,u behind the cell tip). This spatial arrangement suggests an assembly line in which vesicles form near the cell tip and, with progressively increasing distance from the tip, mature by internal lysis and water uptake. The final fusion and liberation of debris into the vacuole is reminiscent of exocytosis (8) with the implication that the vacuole serves as a waste receptacle. (c) Debris-containing vesicles were not seen in vegetative hyphae, but first appeared in the swollen hyphal primordia of very young (0.1 mm) sporangiophores. Thereafter, the vesicle system persisted throughout the development of the sporangiophore and appeared to function after sporulation and maturation of the sporangium. Such early inception of the vesicle system suggests that it is associated with differentiation, i.e., with the switch from hyphal to sporangiophore physiology. Both the early inception and the persistence of the vesicle system past sporulation imply that the vesicle system is concerned more directly with the growth of the sporangiophore than with the process of sporulation. The origin of the cisternae from which debris-filled vesicles form has not been established. However, the common proximity of double-membraned vesicles to nuclei suggests that the precursor cisternae ultimately derive from processes of the nuclear membrane. Alternatively, elements of massed cisternae (which are also perinuclear) may possibly give rise to the vesicles. However, the masses of cisternae themselves presumably originate from the membrane of the adjacent nucleus. In both cases, the common consensus that the endoplasmic reticulum arises from the nuclear membrane (cf. 11) implies that the parental cisternae of the vesicles are nuclear in origin. Similar conclusions have tentatively been voiced in review (9). The suggestion that Golgi vesicles contribute to all cytolytic structures (8) cannot be interpreted easily in this organism since no morphological counterpart of the dictyosome has been seen, but looking at the matter in the other direction, it may be suggested that the vesicle system in the sporangiophore (or some aspect of it) has functions in the sporangiophore analogous to those of the Golgi apparatus in other cells.
PHYCOMYCES I. AUTOPHAGICVESICLES
279
FIG. 13. Cytoplasm of a swollen terminal hypha that has given rise to a sporangiophore primordium of length 0.1 mm. Fie. 14. The initial vacuole of a sporangiophore primordium (same specimen as in Fig. 13), suggesting an origin by the fusion of debris-containing vesicles. Following the completion of this study, Peat and Banbury (17) published electron micrographs of sporangiophores of P. blakesleeanus in which may be seen various autophagic vesicles as described here. These authors cautiously interpreted such structures as "senescent mitochondria" without undertaking a detailed study. This interpretation is clearly superseded by the present findings, in which it appears that the structures seen by Peat and Banbury are complex in origin and by no means consist simply of mitochondria. The author gratefully acknowledges the advice and criticism of Professor K. V. Thimann and of Dr. K. R. Porter and the technical training rendered by Dr. I. R. Gibbons and Mr. R. Branson during the course of this study.
REFERENCES 1. BANBURY,G. H., Encyclopedia of Plant Physiol. XVII/I: 530 (1959). 2. BRACKER,C. E., Pro& Syrup. Colston Res. Soc. 18, 39 (1966).
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ROBERT M. THORNTON
3. BRACKER, C. E. and WILLIAMS, C. M., Proc. 6th Intern. Congr. Electron Microscopy, Kyoto p. 307 (1966). 4. CASTLE,E. S. A m . . ] . Botany 29, 664 (1942). 5. - Science 154, 1416 (1966). 6. DELBR/JCK, M., Ber. Deut. Botan. Ges. 75, 411 (1963). 7. DELBRUCK, M. and REICHARDT, W., in RUDNICK, D. (Ed.), Cdlular Mechanisms in Differentiation and Growth, p. 3. Princeton Univ. Press, Princeton, New Jersey, 1956L 8. DE DUVE, C. and WATTIAUX,R., Ann. Rev. Physiol. 28, 435 (1966). 9. GAHAN, P. B., lntern. Rev. Cytol. 21, 2 (1967). 10. GREHN, J., Jahrb. Wiss. Botan. 76, 93 (1932). 11. HAWKER, L., Biol. Rev. Cambridge Phil. Soc. 40, 52 (1965). 12. MATILE, P., Z. Naturforsch. 20b, 693 (1965). 13. - ibid. 21b, 871 (1966). 14. - Science 151, 86 (1966). 15. MATILE,P. and WIEMKEN,A., Arch. Mikrobiol. 56, 148 (1967). 16. PEASE, D. C., Histological Techniques for Electron Microscopy, 2nd Ed. Academic Press, New York, 1964. 17. PEAT, A. and ]~ANBURY,G. H., New Phytologist 66, 475 (1967). 18. SHROPSHIRE,W. Botan. Rev. 43, 38 (1963). 19. SILVERS,A., Naturwissenschaften 13, 334 (1966). 20. STEWART,K. D. and CUTTER, E. G., Am. ,]-. Botany 54, 632 (1967). 21. THORNTON, R. M., A comparative study of phototropism in Phycomyces and Arena. P h . D . Thesis, Harvard University, Cambridge, Massachusette, 1966. 22. - Plant Physiol. (Proceedings), p. lxviii (1966). 23. - The fine structure of Phycomyces. 1I. The organization of the stage I sporangiophore apex. (In preparation.) 24. THORNTON,R. M. and THIMANN,K. V., J. Cell Biol. 20, 345 (1964). 25. VILLIERS,T. A., Nature 214, 1356 (1967).
s. ULTRASTRUCTURERtSEARCH21, 269--280 (1968)
The Fine Structure of
269
Phycomyces
I. A u t o p h a g i c Vesicles z ROBERT M. THORNTON
Division of Natural Sciences, University of California, Santa Cruz, California 95060 Received August 16, 1967 The fungus Phycomyces blakesleeanus was examined at several developmental stages. The sporangiophores contain vesicles with either a single membrane or a pair of concentric membranes. Such vesicles contain either intact cytoplasm with organelles or a complex form of debris. These findings suggest that certain endoplasmic cisternae of the sporangiophore are engaged in the isolation and lysis of cytoplasm. Longitudinal sections of the sporangiophore reveal axial groupings of vesicles suggesting formation of vesicles near the cell tip, maturation in lower regions, and final discharge of the lytic debris into the large sporangiophore vacuole. The presence of large (above 1 #) granules in vesicles, cytoplasm, and nuclei; the frequent proximity of endoplasmic cisternae to nuclei; the occurrence of nuclei with widely separated inner and outer membranes; and the extension of nuclear membranes into the cytoplasm suggest that the vesicle membranes originate from nuclear membranes. These phenomena were found in all stages of sporangiophore development but not in the vegetative hyphae. Attention was turned in this laboratory a few years ago to the fine structure of the fungus Phycomyces blakesleeanus in hopes of detecting an organelle that could be associated with the sensitive light-growth responses (5) of the sporangiophore. The first result of this work was the detection of "crystal-containing bodies" reminiscent of those in the light-sensitive coleoptiles of Arena (24). A second result has been the finding that the vacuoles of sporangiophores invariably contain debris suggestive of lysed cytoplasm. The origin of this debris is the subject of the present report, and evidence will be presented that the sporangiophore systematically isolates and degrades parts of its own cytoplasm and discards the residues in the vacuole. A similar autophagic system has been reported in the zygomycete GilbertelIa (2, 3), and hydrolytic enzymes are known to occur in vesicular particles in Neurospora (14) 1 This investigation was supported by predoctoral National Science Foundation Fellowships to author and by grant No. GB 3241 from the National Science Foundation to Professor K. V. Thimann. the
270
ROBERT M. THORNTON
and in the vacuoles of yeast cells (15). These recent reports on fungal materials are paralleled by growing evidence for lytic structures in plant cells, either as lysosomelike organelles (12, 19, 25) or as autophagic systems related to the formation of vacuoles (13, 20). However, autophagy is far better known in animal cells; the extensive literature has recently been summarized by Gahan (9) and by de Duve and Wattiaux (8). The consensus expressed in both reviews is that autophagy may turn out to be significant in the control of cellular activities, especially during differentiation. That autophagy should occur in the sporangiophore of Phycomyces bIakesleeanus is of special interest in this regard, because the control of growth in this giant cell has been the subject of very precise studies for half a century (see 1, 5, 6, 18). Indeed, it was proposed a decade ago (7), on quantitative grounds, that the bulk of the "growth precursor" that finds its way into the growing region of the sporangiophore is converted into "waste" and is not used directly in growth. The findings reported here may well bear out the latter prediction and should be useful in subsequent work on the role of autophagy in differentiation. These results have been briefly reported elsewhere (22).
MATERIALS AND METHODS
Phycomyces blakesleeanus, strain G5(+) (collected and identified by Dr. H. E. Gruen) was maintained in pure culture on petri dishes of potato-dextrose-agar (21). The cultures were incubated at temperatures of 13, 19, or 24°C either in complete darkness or with continuous light from overhead laboratory fluorescent lamps. Ctdtures were examined and specimens manipulated with the aid of a Bausch and Lomb binocular dissecting microscope at 15 or 25 diameters. For higher resolution, a Zeiss phase microscope was employed. Specimens were fixed for electron microscopy either by excising hyphae and sporangiophores and immersing them in small vials of fixative, or by flooding entire cultures with fixative and delaying excision until after fixation was terminated. Preliminary studies indicated that cells fixed with permanganate were too brittle to carry through the subsequent stages of preparation. The present results were obtained by means of glutaraldehyde fixation, according to the following technique. Specimens were fixed for 5-13 hours at room temperature in 5% glutaraldehyde in 0.1 M phosphate buffer at pH 6-6.3. After several washes in plain buffer, the specimens were postfixed in 1% OsO~ in a similar phosphate buffer for 2-4 hours at room temperature, washed in distilled water, dehydrated with acetone and embedded in Epon or Araldite (16). The use of propylene oxide in embedding was abandoned after preliminary indications of associated damage to the cytoplasm of the sporangiophores. The resin-infiltrated specimens were arranged in flat aluminum foil boats before polymerization. In the cured blocks, both hyphae and sporangiophores could then be selected and oriented in the microtome chuck with the aid of a dissecting microscope allowing the preparation of longitudinal- or cross sections of the cells. The orientation of specimens was
PHYCOMYCES I. AUTOPHAGIC VESICLES
271
further checked by examination of thick (1 tt) sections under the light microscope after staining with toluidine blue. Thin sections were stained with lead citrate and uranyl acetate (16) and examined with an RCA EMU 3H electron microscope.
OBSERVATIONS Each sporangiophore originates as a lateral bulge on a short, swollen terminal hypha that is club-shaped and packed with yellowish, vesicular cytoplasm (10, 21). The outgrowth of the sporangiophore is accompanied by the initiation and continual expansion of a large vacuole that confines the protoplasm of the sporangiophore to a peripheral layer against the cylindrical cell wall. Before sporulation, the apex of the sporangiophore tapers smoothly to a rounded tip. Of the tapering portion, the uppermost (distal to the mycelium) 100-200 # of the sporangiophore is filled with a mass of cytoplasm. Eventually the extreme apex of the cell balloons into a terminal sporangium within which sporulation occurs. Afterward the sporangiophore continues to elongate for many hours (4). The apical 100-200 # of the sporangiophore was found to be rich in vesicles that are variable in shape, bounded by a unit membrane and filled with debris of a composite granular, membranous and globular character. Such vesicles occur both before (Fig. 1) and after (Fig. 2) sporulation. The sporangiophore vacuole was found to contain debris like that in the cytoplasmic vesicles. While this is true before sporulation (Fig. 3), much more marked accumulations of debris in the vacuole were to be seen after sporulation (Fig. 4). In both stages of development, the vacuolar membrane appears to fuse with many cytoplasmic vesicles. In most such instances debris was found in the attached vesicles as well as the adjacent regions of the vacuole (Figs. 3 and 4). Examination of longitudinal sections revealed an orderly arrangement of debriscontaining vesicles and possibly related structures along the axis of the sporangiophore before sporulation. Several zones have been distinguished, as follows. The lowest region, around the head of the vacuole (termed zone D, 100-200 # below the cell tip) is rich in vesicles that average several microns in diameter (Fig. 3). These vesicles contain loosely packed debris, have a single membrane, and are generally either spherical or cusped as if recently derived by the fusion of a pair of spherical vesicles. The acropetally adjacent region (zone C, 50-150 # in length) contains large numbers of amorphous vesicles measuring about 1 or 2 tt in the largest dimension. In each vesicle a single membrane surrounds a tightly packed mass of debris (Fig. 1). Centered about 25 # from the cell tip is a region (zone B) containing several types
272
ROBERT M. THORNTON
of vesicular structure that will be discussed in detail below in connection with the origin of debris-containing vesicles. Finally, the extreme apex (uppermost 10-20 #) of the sporangiophore (zone A) contains few of the vesicles under consideration here. The cytoplasm of the sporangiophore is in general p o o r in the elongated cisternae that are customarily regarded as endoplasmic reticulum in other organisms. The cytoplasm is rich in small cisternae, however. Groups or masses of cisternae, apparently reticular in character, lined with granules of ribosomal dimensions, were c o m m o n l y encountered close to nuclei in sporangiophores before sporulation (Fig. 10). The perinuclear positioning of these cisternae was c o m m o n enough to suggest that, with appropriate sectioning, each of the m a n y nuclei in the sporangiophore would prove to be associated with a mass of endoplasmic cisternae. Reticulae occur remote f r o m nuclei as well, and after sporulation tracts of reticular cisternae spanning m a n y microns were f o u n d (Fig. 2). T h r o u g h o u t the mycelium and sporangiophore, the cytoplasm contains abundant structures composed of an electron dense, fibrous core surrounded by a single membrane (Fig. 5). Such "dense bodies" appear either circular or rod-like, with dimensions of the order of 0.2-0.5/z. Their nature, origin, and function have not yet been determined.
Key to abbreviations
a c d
autophagic (debris-containing) vesicle endoplasmic cisterna debris db dense body dm debris resembling degraded mitochondrion g granular particle h hyphal vesicle
m mitochondrion n nucleus ng nuclear granule np extension of nuclear membrane t microtubule V vacuole of sporangiophore
The bar on each micrograph represents one micron. All magnifications are approximate. Subject material
Figs. 1, 3, 5-11: sporangiophores of length 0.75M mm, before sporulation Fig. 2, 4: sporangiophores after sporulation Fig. 12: growing tip of a hypha Fig. 13: swollen hypha (nongrowing) from which a sporangiophore has arisen Fig. 14: sporangiophore primordium of length 0.1 mm Fro. 1. A debris-containing vesicle in zone C. Fro. 2. Groups of debris-containing vesicles and endoplasmic cisternae in the growing zone after sporulation. FIG. 3. The tip of the sporangiophore and the adjacent cytoplasm of zone D, illustrating the fusion of vesicles with the vacuole and consequent addition of debris to the vacuole. Note the more swollen appearance of vesicles here than in zone C (Fig. 1). FIG. 4. Vacuole and adjacent cytoplasm of the growing zone after sporulation, showing the large quantities of debris that accumulate in the vacuole.
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ROBERT M. THORNTON
While dense bodies almost invariably occur free in the cytoplasm, occasionally a dense body and its surrounding cytoplasmic matrix were found enclosed within a membranous sac. The sac consisted in some cases of a pair of concentric unit membranes (Figs. 6, 9, and 10), in other cases a single membrane or a compromise involving areas of single and double membrance structure (Fig. 7). Such figures were found chiefly in zone B, though instances occurred in zone C as well. Less commonly, mitochondria with surrounding cytoplasmic matrix were seen enclosed within a membrane sheath (Fig. 8). Primarily in zone B, areas were often encountered in which endoplasmic cisternae partially surround a portion of cytoplasm (Fig. 7). These partial surrounds, as well as the complete cytoplasm-filled sacs described in the previous paragraph, were invariably found near nuclei, though, since zone B is rich in nuclei (23), this may not be significant. In the intact cytoplasm, in the vacuole, and in the lumens of debris-containing vesicles there occurred electron dense, granular particles averaging several microns in diameter (g in Figs. 3, 9 and 13). Sections of these particles are commonly either circular or kidney-shaped (as in Fig. 13) suggesting that the three-dimensional shape often resembles an indented ball. The origin of these particles became a matter of interest when it was found that nuclei sometimes contain particles of similar granulation and electron density (Fig. 11). Occasionally, nuclei were found in which the separation of the inner and outer membrane was irregular (as in Fig. 11). In some cases a space as wide as a micron intervened between the two membranes of the nuclear sheath. The possibility that these separations were artifacts seems to be excluded by the fact that the majority of nuclei had uniformly separated membranes. In contrast to the sporangiophores, no debris-containing vesicles could be found in the vegetative hyphae. Abundant vesicles are present in the hyphal apices, but these invariably contain nothing more than a variety of electron-dense globular material (Fig. 12). Each hypha vesicle possesses a single limiting membrane, and the hyphae exhibit no sacs enclosing cytoplasm or organelles. The absence of debris-containing vesicles in the vegetative mycelium raises a question concerning the point in differentiation at which such vesicles begin to be made. Examining extremely young sporangiophores (e.g., 100 # long; cf. Figs. 13 and 14), FIo. 5. Several dense bodies and fortuitously related microtubules in zone B. FIG. 6. A vesicle in zone B, composed of two membranes surrounding a mass of ground cytoplasm in which a dense body is embedded. Fro. 7. A region of cytoplasm (a) partially surrounded by endoplasmic cisternae. The enclosed plasm contains a dense body and small vesicles typical of cytoplasm (in contrast to nucleoplasm), and the cisternal membranes are more narrowly spaced than the membranes of nuclei. Zone B. F~. 8. A mitochondrion enclosed within a vesicle in zone C.
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the cytoplasm of the swollen parental hypha was found to contain numerous vesicles many of which contain membranous debris (Fig. 13). In these vesicles the debris consists entirely of nested membranes resembling highly distorted mitochondria embedded in fluid space within a surrounding membrane. The primordial sporangiophore apparently owes the first vestige of its vacuole to the fusion of a few such large vesicles containing small amounts of membranous and granular debris (Fig. 14). The electron-dense globular material characteristic of vesicles in the older sporangiophores only begins to appear at a later time, and granular particles of the type that are subsequently prominent in vesicles and vacuole are initially present in the cytoplasm but not in the vesicles of the swollen hypha (Fig. 13). DISCUSSION The evidence bearing on the deposition of debris in the vacuoles of sporangiophores may be divided into several parts for interpretation as follows. (a) Vesicles may be lined with either one or two membranes. In either case, the contents of the vesicle may be either debris or cytoplasm with recognizable dense bodies, large granular particles, or mitochondria. However, in general the vesicles which show only a single membrane are filled with debris, while those with two membranes contain "intact" cytoplasm. Furthermore, regions of cytoplasm are found which are partly surrounded by endoplasmic cisternae. The simplest interpretation for these observations is that certain endoplasmic cisternae come to envelop regions of cytoplasm. If this is true the earliest complete vesicles should possess a double membrane around a mass of intact cytoplasm. Lysis of the contents would follow, together with a reduction from two membranes to one. This proposal matches the mechanism for formation of autophagic vesicles in animal materials, as recently reviewed (8, 9), except that in animal materials the parental cisternae could more clearly be identified as endoplasmic reticulum. It seems appropriate, therefore, to apply the term "autophagic vesicle" to the debris-containing vesicles in the sporangiophore. The validity of this nomenclature is based on the enclosure of organelles within sacs, and no comment can yet be made on the presence of hydrolytic enzymes within the vesicles (cf. 8). However, the presence of hydrolytic enzymes in the vacuoles of higher plant cells (13) suggests a broad uniformity here. FIG. 9. A debris-containing vesicle the double envelope of which clearly consists of a pair of unit membranes. FIG. 10. A nucleus in zone B, showing an extension of the membrane into the cytoplasm. Note also the small group of perinuclear cisternae (c). FI~. 11. A nucleus in zone B, containing a granular particle and having irregularly separated inner and outer membranes. FIG. 12. Cytoplasm about 200/~ behind the apex of a growing hypha of the vegetative mycelium.
°# E
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In addition, the presence of large granular particles in both nuclei and vesicles raises the possibility that some of the debris-containing vesicles might be nuclei in which the observed separation of inner and outer membranes is followed by lysis of the nucleoplasm. This mechanism cannot account for the incorporation of mitochondria and dense bodies into vesicles, however, and does not explain the presence of granular particles in the cytoplasm. (b) Spatially, the sporangiophore apex is organized so that the most apical regions (zone B in particular), contain most of the double-membraned vesicles and instances of partially enveloped cytoplasm. Lower regions contain predominantly single-membraned vesicles filled with debris. Vesicles of the latter type, as judged by the appearance of the contained debris, swell and fuse with the membrane of the large sporangiophore vacuole in zone D (about 100-200 ,u behind the cell tip). This spatial arrangement suggests an assembly line in which vesicles form near the cell tip and, with progressively increasing distance from the tip, mature by internal lysis and water uptake. The final fusion and liberation of debris into the vacuole is reminiscent of exocytosis (8) with the implication that the vacuole serves as a waste receptacle. (c) Debris-containing vesicles were not seen in vegetative hyphae, but first appeared in the swollen hyphal primordia of very young (0.1 mm) sporangiophores. Thereafter, the vesicle system persisted throughout the development of the sporangiophore and appeared to function after sporulation and maturation of the sporangium. Such early inception of the vesicle system suggests that it is associated with differentiation, i.e., with the switch from hyphal to sporangiophore physiology. Both the early inception and the persistence of the vesicle system past sporulation imply that the vesicle system is concerned more directly with the growth of the sporangiophore than with the process of sporulation. The origin of the cisternae from which debris-filled vesicles form has not been established. However, the common proximity of double-membraned vesicles to nuclei suggests that the precursor cisternae ultimately derive from processes of the nuclear membrane. Alternatively, elements of massed cisternae (which are also perinuclear) may possibly give rise to the vesicles. However, the masses of cisternae themselves presumably originate from the membrane of the adjacent nucleus. In both cases, the common consensus that the endoplasmic reticulum arises from the nuclear membrane (cf. 11) implies that the parental cisternae of the vesicles are nuclear in origin. Similar conclusions have tentatively been voiced in review (9). The suggestion that Golgi vesicles contribute to all cytolytic structures (8) cannot be interpreted easily in this organism since no morphological counterpart of the dictyosome has been seen, but looking at the matter in the other direction, it may be suggested that the vesicle system in the sporangiophore (or some aspect of it) has functions in the sporangiophore analogous to those of the Golgi apparatus in other cells.
PHYCOMYCES I. AUTOPHAGICVESICLES
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FIG. 13. Cytoplasm of a swollen terminal hypha that has given rise to a sporangiophore primordium of length 0.1 mm. Fie. 14. The initial vacuole of a sporangiophore primordium (same specimen as in Fig. 13), suggesting an origin by the fusion of debris-containing vesicles. Following the completion of this study, Peat and Banbury (17) published electron micrographs of sporangiophores of P. blakesleeanus in which may be seen various autophagic vesicles as described here. These authors cautiously interpreted such structures as "senescent mitochondria" without undertaking a detailed study. This interpretation is clearly superseded by the present findings, in which it appears that the structures seen by Peat and Banbury are complex in origin and by no means consist simply of mitochondria. The author gratefully acknowledges the advice and criticism of Professor K. V. Thimann and of Dr. K. R. Porter and the technical training rendered by Dr. I. R. Gibbons and Mr. R. Branson during the course of this study.
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3. BRACKER, C. E. and WILLIAMS, C. M., Proc. 6th Intern. Congr. Electron Microscopy, Kyoto p. 307 (1966). 4. CASTLE,E. S. A m . . ] . Botany 29, 664 (1942). 5. - Science 154, 1416 (1966). 6. DELBR/JCK, M., Ber. Deut. Botan. Ges. 75, 411 (1963). 7. DELBRUCK, M. and REICHARDT, W., in RUDNICK, D. (Ed.), Cdlular Mechanisms in Differentiation and Growth, p. 3. Princeton Univ. Press, Princeton, New Jersey, 1956L 8. DE DUVE, C. and WATTIAUX,R., Ann. Rev. Physiol. 28, 435 (1966). 9. GAHAN, P. B., lntern. Rev. Cytol. 21, 2 (1967). 10. GREHN, J., Jahrb. Wiss. Botan. 76, 93 (1932). 11. HAWKER, L., Biol. Rev. Cambridge Phil. Soc. 40, 52 (1965). 12. MATILE, P., Z. Naturforsch. 20b, 693 (1965). 13. - ibid. 21b, 871 (1966). 14. - Science 151, 86 (1966). 15. MATILE,P. and WIEMKEN,A., Arch. Mikrobiol. 56, 148 (1967). 16. PEASE, D. C., Histological Techniques for Electron Microscopy, 2nd Ed. Academic Press, New York, 1964. 17. PEAT, A. and ]~ANBURY,G. H., New Phytologist 66, 475 (1967). 18. SHROPSHIRE,W. Botan. Rev. 43, 38 (1963). 19. SILVERS,A., Naturwissenschaften 13, 334 (1966). 20. STEWART,K. D. and CUTTER, E. G., Am. ,]-. Botany 54, 632 (1967). 21. THORNTON, R. M., A comparative study of phototropism in Phycomyces and Arena. P h . D . Thesis, Harvard University, Cambridge, Massachusette, 1966. 22. - Plant Physiol. (Proceedings), p. lxviii (1966). 23. - The fine structure of Phycomyces. 1I. The organization of the stage I sporangiophore apex. (In preparation.) 24. THORNTON,R. M. and THIMANN,K. V., J. Cell Biol. 20, 345 (1964). 25. VILLIERS,T. A., Nature 214, 1356 (1967).