Concerning the Role of Mitochondria in Cryptobiosis F. Stephen Vogel, MD, Kenneth S. McCarty, Jr., MD, PhD, Doyle G. Graham, MD, PhD, and Lieselotte A. K. Kemper

The metabolic characteristics of the mitochondria of Agaricus bisporu are altered in the zygote by specific inhibitors that permit them to retain structural integrity in the dormant spore and enable them to initiate energy production, with apparent protein synthesis and replication during the initial phase of germination. The insensitivity of the earliest events of germination to selective cytoplasmic and nuclear inhibitors characterizes this as a transient period of unusual mitochondrial autonomv. To define the intrinsic metabolic potentials of the organelle and its role in cryptobiosis, mitochondria were fractionated aseptically from presporulating zygotes and were placed in dialysis chambers surrounded by nutrient media at 15 C. For periods through 48 hours, the isolated mitochondria manifested the capacitv to incorporate labeled amino acids linearly into proteins and retained stable electrophoretic protein profiles for more than 5 days. They maintained fine structural integrity for at least 10 davs. some developed septational membranes, and they increased numericallv. These metabolic activities were dependent upon a nutrient substrate. (Am J Pathol 80:499-518, 1975)

MORE THAN 250 years ago, Leeuiwenhoek noted the existence of dormant, or crvptobiotic, forms of life as spores and seeds, and questioned how metabolicallv active vegetative cells give origin to dormant spores that are devoid of metabolism vet viable and capable of germination even after manv vears.1 Having observed that germination is initiated in many species by simple hvdration, Leeuwenhoek stuggested that the induction of dormancv is effected largelv bv dehvdration.2 Anhvdrobiosis is still emphasized in the induictive phase of the crvptobiotic state.3 In Agaricus bisporus, the fnrit bodv or sporophore develops for the singuilar purpose of sporulation, Spores are formed bv the zvgotes, or basidial cells, on the surfaces of the gills (Figuire 1 ).4 It has been demonstrated that duiring the prodromal period of sponrlation, metabolic inhibitors appear in the gill tissuies. Identified among these are a family of compouinds that consists of a stable precuirsor. 'v-L-glutaminvl-4hvdroxybenzene and two quinoid derivatives that are formed sequentiallv by oxidation of the phenolic precuirsor.5-'0 Althouigh it has been sulggested that the first of these, a pink compound with maximuim light absorbance From the Department of

Pathology,

Duke Universitv Medical Center.

Duirham. North Carolina

Suipported in part by Grant GM-16531 from the National Instituite of General Medical Science. National Instituites of Health Accepted for publication May 8. 1975 Address reprint requiests to Dr F Stephen Vogel. Department of Universitv Medical Center. Durham, NC 27710

Pathology,

Box 3712. Dulke

499

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at 490 nm, is 'Y-L-glutaminyl-3,4-benzoquinone,8'9 uncertainty about its precise molecuilar strticture still remains. Nevertheless, it is clear that this quinoid specifically inhibits several steps in mitochondrial energy prodluction.7 Also, by its strong suilfhydryl binding properties, it sharply cuirtails mammalian DNA polymerases." The second quiinoid notably suippresses ribosomal protein synthesis 13,15 and E. coli RNA polymerase."2 Thus, it would appear that, within the zygote, mitochondrial energy produiction, protein synthesis, and nucleic acid synthesis are sharply cturtailed before mitochondria, ribosomes, and two haploid ntuclei are translocated by cytoplasmic flow from the zygote throuigh the sterigma into each exospore (Figuire 1).6 Evidence from other species indicates that the initial phase of germination is accompanied by reactivation of mitochondrial energy prodluction and by a notable nuimerical increase in mitochondria.'6 These earliest events of germination are insensitive to selective cytoplasmic and nuiclear inhibitors."" Thuis, during a brief period, mitochondrial metabolism apparently proceeds auitonomouisly, seemingly independent of immediate nuclear and cytoplasmic control. The struictuiral characteristics of the fruiit body lend themselves favorably to the sterile isolation of mitochondria from the presporuilating zygotes. Until the onset of sporulation, a veluim stretches from the stipe to the cap, or pileuis, and protects the gills from suirface contamination.4' 6 It is technically possible to fractionate mitochondria aseptically from these gill tissties.1'2' The characteristics of growth and matuiration of the frulit body determine that this mitochondrial fraction will be derived predominently from the germinal cells. The cylindrical mycelia that predominate in the fruiit body are separated one from another by septations, buit each septation contains a prominent pore,4 and enlargement of the mushroom is accompanied by an intercelluilar flow of cytoplasm from the ground mycelia to the gills.22 Duiring the immediate prodromal period of sporuilation, this movement of cytoplasm fluishes mitochondria from the vegetative cells of the gills and concentrates them in the zygotes.6 It has been demonstrated that the respiration and ribosomal protein synthesis of these isolated mitochondria are markedly suippressed by the quinoid inhibitors.6'7"13 It seems logical to assume that in this latent state, these mitochondria have decreased vuilnerability to inijury, as they have during their sojouirn in the spore, and thuis more effectively withstand the trauma of isolation and extracelluilar maintenance. Characteristically, quinoid compouinds are uinstable in aquieouis sollutions. Accordingly, the two derivatives of Y-L-glhtaminyl-4hydroxybenzene are notably labile and are readily oxidized and

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polymerized to inert melanins.7"8 In agreement with Leeuiwenhoek's observations,2 it might be suiggested that hvdration of the spore initiates inactivation of the inhibitors and triggers a return to the vegetative state.6' 17 It might also be hvpothesized that degradation of the mitochondrial inhibitors in vitro wouild release metabolic potentials in the isolated organelle that are normally activated duiring the initial phase of germination. The present stuidies souight information relative to this hypothesis. It was first quiestioned whether the isolated mitochondria had the capacity over prolonged periods to incorporate labeled amino acids into proteins and, secondly, whether the molecuilar profiles of the mitochondrial proteins reflected stabilitv or degradation in nuitrient and nonnuitrient extracelluilar environments. The morphologic correlates were stuidied by electron microscopy. Materials and Methods Agaricus bisporus was grown in the laboratory on compost impregnated with mvcelia by the Mtushroom Supply Company. Touighkenamon. Pa. Abundant mveelial growth and fnrit bodv formation were obtained in environmental chambers at 15 C with high

huimiditv. Preparation of Mithonda Mitochondria were prepared at 0 to 4 C. essentiallv as previouslI described. 19,2122,24 All preparations were made in an area presterilized by uiltraviolet radiation. Soluitions. instniments, and glassware were sterile. Fnrit bodies were harvested prior to nrptlure of the veluim and immediately ice-chilled. The suirface of each velum was flamed briefly-, and the stipe was broken from the pileuis. The veluim was excised. and the gill tisslue removed with sterile blades. Tissuies were homogenized bv a Dotunce homogenizer (Blaessig Glass Co.) 10 ml /g in 0.35 M sucrose containing 0. Ic bovine serum albuimin (BSA). Fifteen strokes each were made bv the loose and tight plunger. The homogenate was centrifuiged in a Beckman No. 21 rotor. 900g for 15 minuites. The sediments were discarded. and the suipernatants centrifuiged at 5.660g in the same rotor for 20 minutes. Each pellet was homogenized in 10 ml of 0.35 M suicrose. withouit BSA, and resedimented in a Beckman No. 40 rotor for 15 minuites at 1.050g. The supematants were decanted and centrifuged in the same rotor for 20 minuites at 6.600g. The mitochondrial pellets were rehomogenized in 0.35 M suicrose so that 1 ml of suispension contained mitochondria from 2g of gill tissuie From pooled samples. measuired voltumes of suispended mitochondria were pipetted into sterile. LKB celltulose dialvsis tuibing with a flat diameter of 9 mm and approximate pore size of 24 nm. First knotted at the lower end. these tuibes were similarlv closed above the suispension. leaving a sizable buibble of air. The tuibes were suibmerged in sterile mediuim contained in graduated cylinders and covered by cotton gauze and aluminum foil; they were then incuibated at 15 C, Media In these stuidies the buiffered mediuim was either natuiral. svnthetic. or suicrose. The natuiral mediuim was prepared according to methods previouslv described 22. 24 To form stock soluition. gill tissuies were homogenized in a Sorvall Omnimixer in 0.35 Nl suicrose. 1

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g/4 ml at 4 C. The homogenate was filtered throuigh filter paper, and the filtrate frozen. To prepare the mediuim, the stock soluition was thawed and dialyzed at 4 C for 24 houirs against five voluimes of a soluition composed of one part of 0.05'M Sorenson's phosphate buiffer, pH 7.0, and two parts of 0.35 M sucrose. Aliquiots of the dialysate were frozen, then thawed, and sterilized by filtration (Nalgene filter; pore size, 0.2 ,u) prior to ulse. The synthetic mediuim was prepared from amino acids (Sigma Chemical Company), minerals, and lipid precuirsors of reagent grade. Its composition was a modification of that of White's mediuim and was patterned after the composition of the natulral meditum as shown by amino acid analysis (Table 1). Penicillin (sodiuim penicillin g, Squiibb) was added to each mediuim in a concentration of 1,000 uinits/ml. Tests for Contamination by Microorganisms Two tuibes of thioglycollate broth, two tuibes of trypticase soy broth and two plates of blood agar were each inoculated with two to three drops of freshly prepared mitochondrial suispension and incuibated at 15 C and 37 C for 5 days. Before each dialysis tuibe was Table 1-Composition of Synthetic Medium L-Alanine L-Asparagine * H20 L-Aspartic acid L-Arginine HCI L-Cysteine

L-Cystine L-Glutamic acid L.Gluatmine Glycine L-Histidine* HCI H9O Hydroxy- L-proline L-lsoleucine L-Leucine L Lysine* HCI L-Methionine

L-Phenylalanine L-Proline L,Serine L-Threonine L-Tryptophan L-Tyrosine L -Valine Calcium chloride * H20 Manganous chloride * 4H20 Magnesium sulfate 7H20 Cupric sulfate 5H20 Ferric chloride * 6H20 Zinc chloride Ammonium molybdate * 4H20 Boric acid KH.2PO4 Na2HPO4 Inositol Choline chloride Sucrose Glucose The pH was adjusted to 7.00. -

-

mg/liter 17.83 26.42 26.62 42.14 24.24 28.06 29.42 29.22 15.02 38.24 26.20 26.24 26.21 36.54 29.84 33.04 23.02 21.02 23.82 40.84 36.24 23.44 16.00 4.00 360.00 2.00 2.00 1.00 1.50 1.50

2,270.00 4,720.00 100.00 10.00 40,000.00 21,000.00

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opened, it was inverted several times to obtain thorough mixing. Fluid from the dialysis tube was drawn into a sterile tuberculin syringe and inoculated into cultuire media as outlined above. Pellets from all suspensions were examined carehfily by electron microscopy as a further screen for bacterial contamination. The demonstration of microorganisms in any specimen eliminated that experiment from the present report. Of more than 200 experiments, less than 10% were contaminated. taioat Amino Add W-C-rporafi It Mft tchonidrW Plow~ Mitochondria isolated and washed four times as outlined for extracellular maintenance were incubated at 15 C in either natural medium, svnthetic medium, or cvtochrome c medium.13 Mitochondria from 2 g of gill tissue were contained in 1 ml. At time intervals of 12, 24, 36, and 48 hours, one dialysis tube was removed from each medium, and the mitochondria were sedimented by centrifugation at 15,000g for 20 minutes, washed and suspended in 0.3 M sucrose, 2 mM EDTA, 30 mM nicotinamide, and Bicine 30 mM, pH 7.5. Incubation was in cytochrome c medium (1 mg mitochondrial protein/ml) with C14 amino acid hydrolysate, 1 juCi/ml (New England Nuclear) for 1 hour. In parallel studies, puromycin (100 #sg/ml) was included in the incubation mixture to evaluate nonspecific isotope absorption.' Aliquots of .1 ml were pipetted onto No. 3 Whatman filter paper discs at intervals of 5, 10, 20, 40, and 60 minutes and quickly dried. The proteins on all discs were simultaneously precipitated by chilled 10% TCA and washed according to the method of Mans and Novelli.-2 Scintillation spectrometry was carried out in a Beckman LS-150 scintillation spectrometer to 2% standard error. Efficiencv for "' C counting was 40% when 15 ml of scintillation mixture per vial was used which contained 0.1 g/liter of 1,4-bis-2-(5phenyloxazolyl-benzene and 4.0 g/liter of 2,5-diphenyloxazole in toluiene. Elthe of MiIochonfit PRoteus Mitochondrial preparations immediately fractionated from gill tissues and those maintained extracellularly for varying periods up to 6 days in natural or synthetic mediuim or in 0.35 M buffered sucrose were washed four times and pelleted by centrifiugation at 15,000g for 20 minutes. The pellet was resuspended in 5 ml of .1 M phosphate bulffer, pH 7.0, and sonicated at 40 W with four 15-second bursts, using a Bronson Sonifier equiipped with a microprobe. This preparation was centrifuged at 300,OOOg for 1 houir in a SW 50.1 rotor. Soluble proteins were precipitated from the supernatant with the addition of six volumes of acetone, washed with ether, air-dried and prepared for electrophoresis according to the methods of Weber and Osborn." The 300,OOOg sediment was treated with 1% sodiuim dodecyl sulfate (SDS) and mercaptoethanol in phosphate buffer, pH 7, and centrifiuged at 30,000g for 30 minutes. The resultant suipernatant was prepared for electrophoresis as above while the insoluble pellet was dissolved in a phenol-formic acid buiffer (phenol: formic acid: water, 2: 1: 1) according to the methods of Braunitzer and Bauer." Electrophoresis was carried ouit in acrylamide gel coluimns, .6 by 15 cm, for 12 houirs at 5 mA/gel, uising methylenebisacrvlamide cross linking at final concentrations of 0.5% bis/10% acrylamide or 0.1% bis/10% acrylamide. Phenol-formic acid-buffered (1:1) gels were polymerized without buffer at a concentration of .075 bis/ 10% acrylamide in 0.6 cm by 15 cm columns. The buffer was equilibrated by electrophoresis at 200 V/gel to a final current of 5 mA. The gel columns (SDS and phenol: formic acid) were fixed in mtltiple changes of 12.5% TCA. The preparations were stained with Coomassie blue and destained in methanol acetic acid. The gels were photographed and then scanned in a Gilford 2400 spectrophotometer equiipped with a linear transport device. ectron Miopy Approximately 1 ml each of freshly prepared mitochondrial suispension and a similar volume from each dialysis tube was used routinely to recover a pellet by centrifiigation

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(No. 40 rotor, 7,900g for 20 minuites). These pellets were fixed immediately in 5% bluffered gluttaraldehyde at 4 C overnight to be postfixed in osmic acid. They were dehydrated in graded strengths of ethanol from 50 to 100% and were embedded in Epon for thin sectioning with a Sorvall Porter-Blutm microtome. The sections were mouinted on 300-mesh copper grids and stained for 30 minuites in lead citrate or 1 % aquieouis uiranyl acetate at 60 C. Counting Mitochondria

This proceduire has been reported previously.21 A known standard of killed Escherichia coli in gluitaraldehyde served as the couinting marker.

Results Amino Acid Incorporation Into Protein by Mitochondria Maintained Extracellularly

The amino acid incorporation into protein by isolated mitochondria maintained extracellihlarly in natuiral mediuim for periods of 12, 24, 36, and 48 houirs is shown in Text-figuire 1. There is continuied incorporation throuigh these periods with persistence of essentially linear rates and with continuied sensitivities to puiromycin (100,ug/ml). In contrast, ribosomes prepared from the cytoplasm of this species incorporated amino acids linearly for less than 40 minuites.13' 27 2.5

25

12 hours

24 hours

'.0 20

0

0

7; E 25 0

20

5

40

60

20

5

40

60

40

60

2548o

36 hours repciey

2,0, 1.5,

05 0

A0 5

20

40

60

5

20

TIME (min)

TEXT-FIGURE 1-Amino acid incorporation by mitochondria maintained extracelluilarly for periods uip to48 houirs. Puiromycin, 100 AtglmI, inhibited to 28.1%, 38.6%, 32.6% and 32.7% at 12, 24,36, and 48 houirs, respectively.

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Protein Profils of Mitodchnria Maintained Extrehularly

The mitochondrial protein components which were soluible in pH 7.0 phosphate buiffer appeared as five major bands in acrvlamide gels (Textfiguire 2). These proteins persisted throuigh 96 houirs of extracellullar maintenance with a quiantitative shift in the region of the third and fouirth major bands (molecuilar weights, 48,000 and 42,000), and there appeared a new protein band at molecular weight 29,000. There was a persistence of the pH 7.0-insoluble, SDS-soluble protein profiles through 120 hours (Text-figure 3). By contrast, there was an appreciable loss of high

Ofhours

Ah TEXT-FIGURE 2-Electrophoretic pattern of protein profiles of pH 7 0 soluble fractions of mitochondria recently fractionated from gill tissues of Agaricus bispos and those maintained 96 hours extracellularv.

I o

I

I

Distance (cm) .

96 hows

506

~

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American Journal of Pathology

molecular weight proteins when mitochondria were incubated in sucrose containing no amino acids (Text-figure 4). Electrophoresis of the SDS-insoluble proteins in phenol-formic acidbuffered gels revealed the presence of two discernible bands 0.7 and 0.5 cm from the gel origin (+). These bands were retained through the second and fourth days quantitatively with a 30% decay of the 0.7 cm band by Day 6. Morphology of Mitochondria Maintained Extracellularly

Many mitochondria in each series that were maintained extracellillarly for periods up to 10 days retained suich basic struictuiral featuires as a duial .A

.

0

S6

A 37,000

60.000 ~~

.3

0

~

.6

hours

,37,0oOOO

60.000

11.700

00

72, 37.000

hou

11.700

0

96 hows

24 ho.n..

~

~

~

-37000000 .6

DISTANCE (em1

~

~

11700

000 1.7700

.0

1170

1,0

3

0L 70

37,000

DISTANCE

11.700

(cm)

TEXT-FIGURE 3-Electrophoresis of pH 7.0 insoluble proteins from mitochondria maintained in natural medium for periods up to 5 days.

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MOLECULAR WEIGHTS 60.000

37000

507

1700

i1

60,000

l700

3X000

TExr-FiGuRE 4-Elec-

trophoretic profiles of pH 7.0 insoluble

pro-

teins of mitochondria maintained for 4 days in

synthetic medium (A), natural medium (B), and buffered sucrose (C).

60,000

31000

1,700

.5

E

0,S,,, L,,S

a

I

DiSTANCE

Ccmj

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peripheral envelope, cristae, a granuilar matrix, DNA filaments, and a general uiniformity in size. In addition, there were morphologic indices of biosynthetic activity: an apparent increase in the nuimber of cristae and the development of septational membranes (Figuires 2-8). Since the transecting membranes differed in their degree of struictutral definition from mitochondrion to mitochondrion, it was quiestioned whether or not these represented stages in a sequience leading to fission. As an approach to the quiestion, the morphologic characteristics of these profiles were evaluated. Mitochondria without septational membranes were regullarly spherical, while those with this membrane were elongated in the axis perpendicullar to the membrane (Figures 4-7). A more definitive struictuiral featuire was the infolding of the inner leaf of the peripheral envelope which luniformly occuirred at contralateral points approximately equiidistant from the two poles of the elongated mitochondrion (Figture 4). In most instances, these invaginations involved only the inner layer of the mitochondrial membrane (Figures 4-7). When the invaginations were clearly defined, a stria of electron-dense material traversed the mitochondrion between them. These striae were predominantly granuilar btit often contained membranouis formations in irreguilar distribuition (Figuire 4). Cristae bordered these striae buit were not noted to cross them. Some transecting membranes had the appearance of ill-defined uinit struictuires (Figuires 4 and 5). Seemingly, these represented an early stage in matuiration, for other more discrete membranes were duial (Figuires 6 and 7). In the latter instance, each constituent leaf of the duial membrane was continuiouis with the inner component of its corresponding half of the peripheral envelope (Figure 7). There was then no distinction in struictuire between the transecting segment of the membrane and the inner component of the original mitochondrial envelope. The ouiter component of the mitochondrial membrane was rarely seen to participate in the process of segmentation (Figuire 8). One wouild expect fractuire of the oluter membrane to be the final event in the separation of the two poles of the mitochondrion; althouigh uinilateral fractuires were noted; these couild not be distinguiished from artifacts. Population Density of Mitochondria Maintained Extracellularly

In close agreement with puiblished observations,21 in three stuidies in which mitochondria were sustained by synthetic nuitrient mediuim and counted daily for 10 days, there was a variable but patterned ntumerical response. In Series 1, a Day 0 concentration of 12 X 1015/ml increased progressively to 22.1 X 1015/ml on Day 4, and this was followed by a

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gradual decline to 14.6 X 1015/ml on Day 10. Series 2 showed an increase from 3.4 X 10'5/ml on Day 0 to 10.2 X 1015/ml on Dav 3, followed bv a progressive decline to 3.6 X 10'5/ml on Day 10. In Series 3, a Day 0 concentration of 5 X 1015/ml increased sharply to 10.8 X 105/ml bv Day 1 and then gradually declined to 1.7 x 10'5/ml on Day 10. The nuimber of mitochondria with septational membranes was appreciably greater in Series 1, and in all series, were more ntumerouis duiring the first 5 davs. However, septational membranes were noted throuighouit the period of studv in all series (Figures 3-5). Dicussion By themselves, the profiles of elongated mitochondria with septational membranes are static figures in an unnatuiral environment. The possibilitv exists, however, that these forms represent points in a sequence of events that are analagouis to those that occtur intracelluflarlv. This is underscored by the close morphologic similarity of the extracelluflar mitochondria profiles to those of mitochondria that have been observed intracellularly under natural and experimental conditions. For example, it has been observed by electron microscopy that at the time of metamorphosis from puipal to adult stage, verv few mitochondria are present in the cells of the fat body of the insect Calpodes ethlius." Duiring the first dav of adult life there is a rapid increase in the nuimber of mitochondria per cell, and morphometric analyses of these organelles provide evidence of mitochondrial growth and division. Duiring this brief interval, the structural transformations in the mitochondria that suiggestively lead to the formation of partitioning membranes and fission, are indistinguiishable from those noted in the isolated muishroom mitochondria. Similar transformations have been observed over a more prolonged period in rat liver. Following the administration of the aminoazo dve, 2-Me-DAB, a noncarcinogenic hepatotoxin, notable increases occur in the number of mitochondria per liver cell. Manv of these mitochondria are characterized by the presence of a "median douible membrane continuious with the inner limiting membrane of the mitochondrial envelope.'29 These observations paralleled those in mice that had been deprived of riboflavin for 6 to 8 weeks and then were administered this vitamin intraperitoneally. The hepatic mitochondria that had enlarged in the deficient animals promptly developed septational membranes after the administration of riboflavin. These membranes were visible in mitochondria within the intact cell and remained so when this organelle was fractionated in sucrose. Thev were stnretuired in-

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distinguishably from those in the mushroom mitochondria.30 In two neoplasms, oncocytomas of the human parotid gland, cells with a large number of mitochondria showed many with septations, while fewer septations were seen in mitochondria of cells with lesser concentrations of this organelle.31 Although these observations have been made in widely divergent species, the structuiral features of mitochondrial septation and apparent fission are notably similar. It has been suggested that the mitochondrial septational membrane is a modified crista.28 However, the cristae of the muishroom mitochondria, as is true of most plants, are cylindrical in shape, whereas the partitioning membrane must have a broad lamellar configuiration. In addition, similarities exist between the mitochondrial septations and those that have been noted in a few species of chloroplasts,32 an organelle withouit cristae. There is abundant evidence to indicate that the transcriptional and translational capacities of mitochondria in most biologic systems are limited to relatively few proteins.3337 The divergence from the norm by mitochondria from the zygotes of Agaricus therefore, requiires explanation. Previous studies have disclosed that the physical properties of the mitochondrial rRNAs and DNA of Agaricus 13-15 resemble those of Neurospora crassa and other species that occtupy the lower strata of the phylogenetic scale.33-35 In Kleinschmidt preparations, the mitochondrial DNA of Agaricus, released by osmotic shock and puirified on CsCl or ethidium bromide-CsCl gradients appears as linear strands uip to 25 ,u in length and circular forms 2.2 to 5.2 ,u in circuimference.13' 15 Nevertheless, notable differences exist in cell structuire and in the life cycles of Agaricus and Neurospora. In each, the vegetative cells form cylindrical hyphae and are delimited by septations. These contain septal pores that permit the flow of cytoplasm with the transport of mitochondria from cell to cell. The vegetative cells of each species are dikaryotic. The septal pores of Neurospora, but not those of Agaricus, permit the translocation of nuiclei from cell to cell. Thus, there is a more transient spacial relationship between nuiclei and mitochondria in Agaricus, and perhaps a greater degree of mitochondrial fuinctional independance even in the vegetative cells. The significance of this difference is not clear and perhaps it is trivial. However, with sportulation the life cycle of Agaricus deviates greatly from that of Neurospora. The evidence, althouigh still largely circuimstantial, persuiasively suggests that duiring the prodromal period of sporulation the mitochondria in the zygotes of Agaricus are specifically modified to achieve two widely divergent objectives. First, to maintain integrity in the spore for many years withouit active metabolism and, secondly, to initiate energy production and self-replication duiring the

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earliest phase of germination which is manifestlv independent of immediate cytoplasmic or nuclear participation. The first property appears to result from the direct effects of specific inhibitors. Conceptuiallv. degradation of these inhibitors would initiate germination. The source of the metabolic potentials expressed by the mitochondria duiring the initial phase of germination remains unclear; however. the present stuldv provides an experimental model to approach this problem. It seems likely that the biologic principles which govern events as dvnamic and precise as those of crvptobiosis might have therapeuitic application.'-' Note A correction of the molar coefficient based on data given in Arch Biochem Biophys 149:541, 1972 revealed that the actual concentrations of the inhibitors 490 and 360 used in the studies reported in Am J Pathol 76:165, 1974 and 78:33, 1975 were 250 times smaller than the quantities cited. References 1. Leeuwenhoek A van: On certain animalecules fotund in the sediment in guitters of the roofs of houses (Letter 144). The Select Works of Antony Van Leeuwenhoek. Translated bv Samuel Hoole 1798, 4° 2 vols London. 1702 2. Keilin D: The problem of anabiosis or latent life: Historv and culrrent concepts. The Leeuwenhoek Lecture 1958. Proc R Soc Lond [Biol] 150:149-191. 1959 3. Anhvdrobiosis, Benchmark Papers in Biological Concepts. Edited by JH Crowe, JS Clegg. Stroudsbuirg, Pa, Dowden, Huitchinson and Ross. Inc, 1973 4. Vogel FS: Stnretural and functional characteristics of deoxvribonuicleic acid-rich mitochondria of the common meadow mushroom. Agarscus campestris. I. Intracelluilar environment. Lab Invest 14:1849-1867, 1965 5. Weaver RF, Brvne WL, Vogel FS: Formation of the dormant spore in the common meadow mushroom: Appearance of respiratorv inhibitor(s). Fed Proc 27:248. 1968 6.

7. 8.

9. 10. 11.

(Abstr) Vogel FS, Weaver RF: Concerning the induiction of dormancv in spores of Agaricus bisporus. Exp Cell Res 75:95-104, 1972 Weaver RF, Rajagopalan KV, Handler P: Mechanism of action of a respiratory inhibitor from the gill tissues of the sporulating common mushroom. Agaricus bisponrs Arch Biochem Biophvs 149:541-548, 1972 Weaver RF, Rajagopalan KV, Handler P. Jeffs PW, Byrne WL. Rosenthal D: Isolation of Y-L-glutaminvl4-hvdroxvbenzene and Y-L-glutaminvl-3,4-benzoquiinone: A natuiral sulfhvdrvl reagent, from sponulating gill tissue of the mushroom Agaricus bisporus. Proc Natl Acad Sci USA 67:1050-1056, 1970 Weaver RF, Rajagopalan KV. Handler P. Rosenthal D. Jeffs PW: Isolation from the muishroom Agancus bisporus and chemical svnthesis of -Y -L-ghltaminyl-4hvdroxvbenzene. J Biol Chem 246:2010-2014, 1971 Weaver RF, Rajagopalan KV, Handler P. Bvrne WL: Y-L-glutamin%1l-3A4benzoquinone: Struictural studies and enzvmatic svnthesis J Biol Chem 246:2015-2020, 1971 Graham DG, Tve R, McCartv KS Jr, Kemper LAK. Vogel FS: Mechanisms of action

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12. 13. 14.

15. 16. 17.

18. 19.

20. 21. 22. 23. 24.

25.

26. 27. 28. 29.

30. 31. 32. 33. 34.

VOGEL ET AL

American Journal of Pathology

of antibiotic and antineoplastic quinoids from Agaricus bisporus. Am J Pathol 78:43a, 1975 (Abstr) Vogel FS, McGarry SJ, Kemper LAK, Graham DG: Bacteriocidal properties of a class of quiinoid compouinds related to sporuilation in the muishroom, Agaricus bisporus. Am J Pathol 76:165-174, 1974 McCarty KS Jr: The Ribosomes in Evoluition: A Comparison of Mitochondrial and Cytoplasmic Ribosomes. PhD Thesis, Duike University, Department of Pathology, 1973 McCarty KS Jr, Vogel FS, Kinney TD: Comparison of cytoplasmic and mitochondrial ribosomes. In Vitro 8:426, 1973 McCarty KS Jr, Weaver RF, Kemper L, Vogel FS: Properties of isolated mitochondria of Agaricus bisporus: Their relationship to dormancy and the germination of spores. Elect Microsc Soc Am 1973 (Abstr) Suissman AS, Halvorson HO: Spores, Their Dormancy and Germination. New York, Harper and Row, 1966 Suissman AS: The dormancy and germination of fuingus spores. Symp Soc Exp Biol 23:99-121, 1961 Steinberg W, Halvorson HO, Keynan A, Weinberg E: Timing of protein synthesis duiring germination of spores of Bacillus cereus strain T. Natuire 208:710-711, 1965 Vogel FS, Kemper L: Struetuiral and functional characteristics of deoxyribonucleic acid-rich mitochondria of the common meadow muishroom, Agaricus campestris. II. Extracelluilar cuiltuires. Lab Invest 14:1868-1893, 1965 Vogel FS, Kemper L: Intrinsic ribonuicleic acid synthesis in mitochondria of Agaricus campestris uinderscoring the probability of an extranuclear genetic system. Exp Cell Res 47:209-221, 1967 Vogel FS: Meadow muishroom provides a model for the stuidy of mitochondrial DNA. Fed Proc 28:1820-1824, 1969 Bonner JT: The growth of muishrooms. Sci Am 194:96-106, 1956 McCarty KS Jr, Kemper L, Vogel FS: Association of radioactive amino acid label with protein in the absence of peptide synthesis. Anal Biochem (In press) Mans RJ, Novelli GD: Measuirement of the incorporation of radioactive amino acids into protein by a filter paper disk method. Arch Biochem Biophys 94:48-53, 1961 Weber K, Osborn M: The reliability of molecuilar weight determinations by dodecyl suilfate polyacrylamide gel electrophoresis. J Biol Chem 244:4406-4412, 1969 Braunitzer G, Bauer G: UJber die Anzahl der Proteinkomponenten im Lamellarsystem der Chloroplasten. Natuirwissenschaften 54:70, 1967 McCarty KS Jr, Kemper L, Vogel FS: Protein systhesis by mitochondria maintained extracelluilarly In Vitro 7:278, 1972 (Abstr) Larsen WJ: Genesis of mitochondria in insect fat body. J Cell Biol 47:373-383, 1970 LaFontaine JG, Allard C: A light and electron microscope study of the morphological changes induiced in rat liver cells by the azo dye 2-Me-DAB. J Cell Biol 22:143-172, 1964 Tandler B, Erlandson RA, Smith AL, Wynder EL: Riboflavin and mouise hepatic cell structuire and function. II. Division of mitochondria during recovery from simple deficiency. J Cell Biol 41:477-493, 1969 Tandler B, Huitter RVP, Erlandson RA: Ultrastruictuire of oncocytoma of the parotid gland. Lab Invest 23:567-580, 1970 Gantt E, Arnott HJ: Chloroplast division in the gametophyte of the fern Matteuccia struthiopteris (L) Todaro. J Cell Biol 19:446-448, 1963 Rabinowitz M, Swift H: Mitochondrial nuicleic acids and their relation to the biogenesis of mitochondria. Physiol Rev 50:376-427, 1970 Borst P: Mitochondrial nuicleic acids. Ann Rev Biochem 41:333-376, 1972

Vol. 80, No. 3

MITOCHONDRIA IN CRYPTOBIOSIS

513

September 1975

35. Luck DJL: Formation of mitochondria in Neurospora crassa: A qllantitative radioautographic study. J Cell Biol 16:483-499, 1963 36. Ashwell M, Work TS: The biogenesis of mitochondria. Ann Rev Biochem 39:251-290, 1970 37. Coote JL, Work TS: Proteins coded bv mitochondrial DNA of mammalian cells. Euir j Biochem 23:564-574, 1971 38. Vogel FS, Kemper LAK, McGarry SJ, Graham DG: Cvtostatic, cytocidal and potential antitumor properties of a class of quinoid compounds, initiators of the dormant state in the spores of Agancus bisporus. Am J Pathol 78:33-46, 1975 The authors wish to express their deep appreciation to Mr Bernard Ulovd for his valuiable contribuitions, partictularly in the area of electron microscopy

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Figure 3-Higher magnification of mitochondria, that had been maintained extracellularly for 8 days shows well defined dual peripheral membranes, densely granular matrix and cristae that appear more numerous than in tissue mitochondria. 4(X 58,000) Figure 4-As exemplified in this preparation of mitochondria maintained extracellularly for 8 days, the mitochondrion that has a septational membrane is slightly elongated in its 900 axis. Note that the inner layer of the mitochondrial envelope is invaginated and that between these approximately symmetrically positioned invaginations there is an electron-dense stria. This contains granules and membranes with varying degrees of structural definition. (x 62,000)

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7-With total cleavage, the septational membrane bisects the mitochondrion. Note that each leaf is continuous with the inner layer of the peripheral envelope. (X 96,000) Figue 8-The invagination rarely involves both layers of the peripheral envelope thus constricting the mitochondrion in hourglass fashion (X 96,000)

518

VOGEL ET AL MITOCHONDRIA IN CRYPTOBIOSIS

[End of Article]

American Journal of Pathology