the role of the fibrillar component of the surface sheath in the morphogenesis of dictyostelium...

DEVELOPMENTAL BIOLOGY 56, 184-194 (1977)

The Role of the Fibrillar Component of the Surface Sheath in the Morphogenesis of Dictyostelium discoideum

HUD FREEZE AND WILLIAM F. LOOMIS

Department of Biology, University of California at San Diego, La Jolla, California 92093

Received August 6, 1976; accepted in revised form October 29,1976

We have isolated a fibrillar component of the surface sheath ofDictyostelium discoideum by virtue of its insolubility in 9 M urea-2% sodium dodecyl sulfate (US). The US-insoluble material is primarily composed of cellulose, but also contains other carbohydrate components, protein, and lipid. Evidence is presented that the US-insoluble material is a component of the sheath. Sheath isolated from mutant strains lacking the developmentally regulated N-acetylglucosaminidase (NAG), cu-mannosidase, or /3-glucosidase activities is similar in composition to sheath isolated from wildtype strain. Strains lacking NAG are unable to migrate normally. This may result from the markedly lower crystallinity of the cellulose in the US-insoluble sheath isolated from these mutants. Strains lacking a-mannosidase or p-glucosidase migrate normally and the crystallinity of the sheath cellulose is not significantly below that of the wildtype. The correlation between the lower crystallinity of cellulose and the inability of strains lacking NAG to migrate suggests that crystallinity is physiologically important and that the degree of crystallinity is controlled by an enzymatic, mutable process. Strains Ul and UNl, which have ~1% of the wildtype activity of uridine diphosphate glucose pyrophosphorylase activity, develop in a morphologically normal fashion to the slug stage. However, they cannot form stalk or spore cells, nor can they produce cellulose. These strains do not produce any detectable US-insoluble sheath and are fragile and unable to migrate. A continuous, nonfibrillar sheath surrounds the aggregates and is sufficient for normal morphogenesis up to the slug stage. The librillar component gives the aggregate the added rigidity required during migration.

INTRODUCTION

A surface sheath first surrounds aggregates of Dictyostelium discoideum near the end of the aggregation phase of development and functions to integrate the aggregate and limit its size by preventing the entry of new cells (Raper, 1935). When the aggregate transforms into a migrating pseudoplasmodium (slug), new surface sheath is continually synthesized as the cells move through it, leaving the old sheath behind (Shaffer, 1965). After a vari- able period of migration, the slug stops and constructs a fruiting body.

ment (Garrod, 1969; Gerisch, 1960; Shaf- fer, 1962; Takeuchi and Yabuno, 1970; Whitfield, 1964). For instance, cells allowed to develop under water will form only loose piles of cells which do not pos- sess a sheath and which cannot proceed beyond this point in development as long as the cells are submerged. Calculations and observations have suggested that the sheath around the tip of a slug is probably a “viscous liquid” which “congeals” to form a solid, rigid sheath in the region behind the tip (Garrod, 1969; Francis, 1962). The nature of this maturation is not under- stood.

The importance of surface sheath and its Although the surface sheath is impor- role in development have been discussed tant for the proper development of the by Loomis (1972). The sheath appears to be slime mold, there has been little definite essential for development, since conditions information about the chemical composi- which either disrupt its integrity or pre- tion of the material, since it has been diffr- vent its formation block further develop- cult to isolate sufficient quantities for

184

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FREEZE AND LOOMIS Surface Sheath of D. discoideum 185

chemical analysis. Previous micrographic and histochemical qualitative analyses on individual slugs have suggested that the sheath may contain protein, cellulose, and other polysaccharides (Loomis, 1972; Gar- rod, 1969; Farnsworth and Loomis, 1975; Raper and Fennel, 1952; Hohl and Jehli, 1973).

We have isolated a cellulose-containing component from slugs and have presented an analysis of its composition and structure (Freeze and Loomis, 1977). Here we wish to present the evidence that the material we isolated is a sheath component.

Structural gene mutations affecting some developmentally controlled enzymes have been isolated and partially characterized (Dimond et al., 1973; Free and Loomis, 1975; Dimond et al., 1976; Dimond and Loomis, 1976). a-Mannosidase, p-glucosidase, andN-acetylglucosaminidase are all preferentially excreted during axenic growth and development (Every and Ash- worth, 1973) and might have a significant function in sheath metabolism. The surface sheath contains mannose, glucose, and N-acetylglucosamine, which could serve as substrates for these enzymes, and it was, therefore, of interest to see whether or not the composition of the surface sheath was modified in the mutant strains. Mutants lacking p-glucosidase were found to be self-complementary in the macrocyst mating system, which usually requires the involvement of two differ- ent mating types (Dimond and Loomis, 1976). No altered phenotype was found for those mutants lacking a-mannosidase. Mutants which lack N-acetylglucosaminidase (NAG) form small slugs which migrate abnormally (Dimond et al., 1973).

Mutants lacking uridine disphosphate glucose pyrophosphorylase (UPP), which is responsible for the synthesis of uridine diphosphate (UDP)-glucose from uridine triphosphate and glucose l-phosphate, have also been isolated (Dimond et al., 1976). UDP-glucose is probably the imme- diate precursor for cellulose, glycogen, tre-

halose, and mucopolysaccharides (Nikaido and Hassid, 1971). Mutants lacking this enzyme proceed through development up to culmination, but they are arrested at that point and do not form either stalk or spore cells. The stalk, spore coat, and surface sheath all contain cellulose (Raper and Fennel, 1952; Hemmes et al., 1972; Gezelius and Ranby, 1957; Roseness and Wright, 1974; Freeze and Loomis, 1977). Strains lacking UPP are unable to produce the large amounts of cellulose found in stalks and spores, but it is not known whether or not they can produce the small quantities of cellulose present in the surface sheath.

The results presented below indicate that the chemical composition of the 9 M urea-2% sodium dodecyl sulfate (US)-insoluble surface sheath in mutants lacking a-mannosidase, p-glucosidase, and N-acetylglucosaminidase is essentially the same as that in the wildtype. However, the crystallinity of the cellulose, which is often correlated with the strength of cellulose, is considerably below normal in the migra- tionally feeble mutants which lack N-acetylglucosaminidase. Mutants lacking UPP do not have any detectable cellulose-containing component of surface sheath, but are surrounded by another chemically un- characterized, nonfibrillar, continuous component of sheath previously described by Farnsworth and Loomis (1975).

METHODS

Strains. Strain A3 was used as the wildtype. WL4a is an axenic mutant strain which fails to aggregate when placed under developmental conditions. Strains DBL 211 and DBL 230 are derived from A3 and lack the developmentally regulated enzyme N-acetylglucosaminidase (Di- mond et al., 1973). Strain G4 is a mutant strain lacking p-glucosidase activity which was derived from strain A3 (Di- mond and Loomis, 1976). Strains Ml and M2 lack a-mannosidase-1 activity and

186 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

were also derived from A3 (Free and Loomis, 1975). Strain Ul was derived from wildtype strain A3 and possesses <l% of the wildtype uridine diphosphate glucose (UDPG) pyrophosphorylase activity. Strain UN1 was derived from strain DBL 211 and possesses 1% of the wildtype activity of UDPG-pyrophosphorylase (UPP). All strains lacking UPP were grown in association with Klebsiella aerogenes. All other strains were grown axenically in HL-5 medium.

Isolation of the urea-SDS-insoluble surface sheath and trails. The isolation of the urea-SDS (US)-insoluble component of sheath was done according to Freeze and Loomis (1977). Cells were developed to the appropriate stage on large agar trays and harvested in 9 M urea-2% SDS. The lysate was vigorously stirred over a 2-day period and repeatedly washed with US and water. The insoluble material was heated in US, water, and 95% ETOH and lyophi- lized. Isolation of the US-insoluble component of pseudoplasmodial trails was done in a manner similar to that described by Freeze and Loomis (1977).

Chemical assays. Cellulose was deter- mined by the method of Corbett (1963). Amino acid analyses, lipid analyses, and gas-liquid chromatography (GLC) of car- bohydrates were described by Freeze and Loomis (1977).

Microscopy. All light microscopy was done using a Zeiss compound microscope with phase-contrast optics. Electron microscopy was performed on a Phillips 300 by Dr. Paul Farnsworth. Transverse sections were prepared from material fixed in 2% glutaraldehyde in the presence of ru- thenium red (Farnsworth and Loomis, 1975).

RESULTS

Appearance of the US-Insoluble Material

We have isolated material from pre-slug aggregates, migrating slugs, and trails which is insoluble in 9 M urea-2% SDS

(US) (Fig. 1). When the insoluble material is prepared from cells which are developed to the beginning of the slug stage (about 16 hr), the insoluble material appears as large, thin, nearly transparent sheets. The area of some of these pieces is nearly equal to the surface area of an average-sized slug (Fig. 1C). The sheets are smaller if they are isolated from earlier aggregates that are just beginning to accumulate a surface sheath.

Hohl and Jehli (1973) detected cellulase- sensitive, cellulose-sized (lo-15 nm in diameter) fibrils of indeterminate length in the trails left behind migrating slugs. Fi- brils of similar diameter (9-12 nm) are visible in negatively stained preparations of the US-insoluble material isolated from slugs (Loomis, 1975). Data we presented elsewhere show that cellulose is the pre- dominant component in the US-insoluble material (Freeze and Loomis, 1977).

Developmental Kinetics

Figure 2 shows the developmental kinetics of the accumulation of the US-insoluble material. No US-insoluble material could be isolated from the lysate of loll vegetative cells, and there is no isolatable US- insoluble material prior to about 8-10 hr of development when a visible sheath first appears (Raper, 1935; Shaffer, 1965). The amount of isolatable US-insoluble material begins to increase at 8 hr, continues to accumulate until about 12-13 hr of development, and then remains fairly constant for the next 5 hr or so. At this point, the US-insoluble material constitutes about 0.03% of the dry weight of the cells. The rate of accumulation of US-insoluble material increases dramatically at the onset of migration and this high rate of accumulation is maintained as long as the slugs continue to migrate. These kinetics are consistent with those expected for a component of the sheath (see Discussion). Strain WL4a, which fails to aggregate or form a visible surface sheath, does not produce any US-insoluble material when


FIG. 1. Photomicrographs of trails, US-treated trails, and US-insoluble material. (A) Photomicrograph of a trail removed from the rear of a migrating pseudoplasmodium. (B) Photomicrograph of a trail isolated after US treatment as described under Methods. (C) Photomicrograph of US material isolated as described under Methods. (All were viewed using phase contrast optics.)

188 DEVELOPMENTAL BIOLOGY VOLUME 56. 1977

32w -

2 2two - B 0 2400 - 0 ; 2000 - n

g 1600 -

T 1200 -

800 -

FIG. 2. Developmental kinetics of accumulation of US-insoluble material. Vegetative cells were placed on 2% agar at 0 hr and allowed to develop to the stage or time indicated. US-insoluble material was isolated from 5-10 x 10”’ cells at each time point.

placed under developmental conditions for 17 hr.

Composition of the US-Insoluble Material of Wildtype, Mutant Strains and Trails

The composition of the US-insoluble material is presented in Table 1. The detailed analysis of each of these components was presented elsewhere (Freeze and Loomis, 1977). Cellulose comprises a majority of the dry weight of the US-insoluble material, and protein is present at about 15% of the dry weight. Another carbohydrate component called a heteropolymer accounts for about 370, and fatty acids occur in amounts varying from 3-5% of the dry weight. Sulfate comprises about 1%.

US-insoluble material was isolated from mutant strains lacking cY-mannosidase, p- glucosidase, and N-acetylglucosaminidase and compared to the wildtype. The sheath of all the strains tested had 5560% cellulose and contained 15% protein of a similar amino acid composition to that found in the US-insoluble material of A3. Gas-liquid chromatographic analysis of the samples of US-insoluble material isolated from mutants strains indicated that the composition of a heteropolymer isolated from each of the mutants is similar to that of

the wildtype (Table 2). Strains lacking (Y- mannosidase or p-glucosidase contained fatty acids which were similar to the wildtype. Strains lacking NAG were not as- sayed for fatty acids.

Trails left behind migrating slugs also contain cellulose, protein, and a heteropolymer similar to that found in the US- insoluble material (Freeze and Loomis, 1977).

Crystallinity of Cellulose

Cellulose exists as highly crystallized regions dispersed among noncrystalline

TABLE 1

COMPOSITION OF THE UREA-SDS (US)-INSOLUBLE

MATERIALS

Component Maximum percent- age of dry weight of

sheath

Cellulose 65 Protein 15

Heteropolysaccharide 3 Fatty acids 5

Sulfate 1

a The quantitation of each component was deter- mined by the methods of Freeze and Loomis (1977). This table summarizes data previously presented in detail (Freeze and Loomis, 1977).

TABLE 2

SUGARS SOLUBILIZED FROM US-INSOLUBLE MATERIAL BY A %HR METHANOLYSIS~

Sugar Strain (pg/mg of US-insoluble material)

Fucose Xylose Mannose Galactose Glucose N-acetyl

glucosa- mine

Wild- G4 Ml and DBL type M2b 211

2.1’ 2.0 2.0 1.7 1.7 0.7 1.3 2.4

14.9 12.8 15.5 15.5 65 3.4 3.8 4.7

81.0 150.0 125.0 200.0 7.0 4.5 6.0 6.2

(2 Samples were methanolyzed and analyzed by GLC (Freeze and Loomis, 1977). Each number repre- sents the average of at least three determinations which were found to vary less than 10% from each other.

b The values for the two strains listed were about the same, and the two values were averaged.

c From Freeze and Loomis (1977).


(amorphous) regions (Nickerson, 1950; Tripp, 1971; Mann, 1963). The measurement of the amount of crystallinity of cellulose has been the focus of considerable interest, since many of the physical and chemical properties of cellulose are strongly influenced by the degree of crystallinity (Howsman and Sisson, 1954; Wakeman, 1954; Nickerson, 1950; Tripp, 1971; Hermans, 1949). Tensile strength, rigidity, and hardness usually increase with increasing crystallinity, while chemical reactivity, flexibility, and elongation usually decrease with increasing crystallinity. The noncrystalline areas are acces- sible to a variety of degradative reagents, but the crystalline regions are relatively inaccessible to these reagents and are bro- ken down very slowly.

The kinetics of methanolysis of samples provide a convenient way of assaying the relative crystallinity of the cellulose frac- tion (Mann, 1963). Figure 3 shows the kinetics of methanolysis of the cellulose of the US-insoluble material isolated from wildtype strains and strains lacking CX- mannosidase, p-glucosidase, and N-acetylglucosaminidase, together with wood CZ- cellulose as a standard. About 99% of the glucose present in the US-insoluble material is cellulose. Therefore, the kinetics of cellulose degradation are measurable by the rate of solubilization of glucose. The initial rapid release of glucose is due to the preferential attack of the acid upon the noncrystalline regions and the later points show the slower degradation of the relatively inaccessible crystalline regions. The amount of crystalline cellulose may be de- termined by extrapolating the line drawn through the late time points back to they- axis. Thus, a-cellulose is about 88% crystalline, or 12% noncrystalline (120 pg/lOOO pg), and the crystallinity of cellulose in the US-insoluble material from strain A3 is about 85%. The crystallinity of the cellulose from strains lacking acetylglucosaminidase was lower than that of the wildtype (Table 3).

5 10 15 20 25 39 35 40 45 50 55 60 65 70 75

Time (Hours1

FIG. 3. Kinetics of methanolysis of US-insoluble material. About 1 mg (weighted to +lO pg) of various samples from migrating slugs was methanolyzed. At each time point, the acid-insoluble material was sedimented by centrifugation and the superna- tant was removed for analysis. The pellet was washed with methanol and resuspended in fresh methanolic-HCl, and the methanolysis was contin- ued. Each sample was analyzed by gas-liquid chromatography and the results are expressed as micro- grams of glucose solubilized per milligram of cellulose. O-O, wood a-cellulose; U-----O, US-insoluble material of strain A3; O-O, US-insoluble material of strain Ml; A-----A, US-insoluble material of strain G4; O-----O, US-insoluble material of strain DBL 239 from slugs about to migrate.

Lack of Fibrillar Component in Mutant Strains

No US-insoluble material which resembles the sheets isolated from wildtype aggregates could be isolated from up to 2 x

1O’O cells of strains Ul or UN1 allowed to develop for 18 hr, as judged by microscopic observation of the total insoluble material. These strains lack UDPG-pyrophosphorylase and are unable to form cellulose.

The circular profiles normally seen in the surface sheath region of the wildtype are absent in these mutants (Fig. 4). It would appear that the circular profiles compose at least part of the US-insoluble material.

DISCUSSION

We have isolated material which is insoluble in a mixture of urea and SDS. The evidence presented here strongly suggests

FIG. 4. Electron photomicrograph of the surface sheath. (A) Transverse section of the surface of an intact slug of strain A3 (wildtype) showing the lo- to 15-nm-diameter circular profiles (arrow) apposed to the continuous surface sheath. (B) Transverse section on an 1%hr aggregate from mutant strain Ul which lacks UDPG-pyrophosphorylase activity. Note the absence of circular profiles.

190


TABLE 3

RELATIVE CRYSTALLINITIES OF CELLULOSE OF THE

US-INSOLUBLE MATERIAL FROM WILDTYPE AND

MUTANT STRAIN@

Strain Stage of develop- Relative ment crystallinity

A3 Tipless aggregate 72.9 f 2.9 A3 Tipped aggregate 76.0 A3 Migrating slug 84.1 ? 3.0 M2 (o-MAN-1 Migrating slug 77.0 G4 (/3-GLU-) Migrating slug 78.0 DBL 239 (NAG-) Migrating slug 60.0 DBL 211 (NAG-) Migrating slug 70.0 cr-Cellulose 89.0 5 2.0

n The kinetics of methanolysis of samples were used to determine crystallinity of cellulose as described in the legend of Fig. 3. The crystallinities were calculated by extrapolating late time points to the y-axis. Values without standard deviations are the result of single determinations.

that this material is at least one of the components of the surface sheath of D. discoideum. The US-insoluble material can be isolated as thin sheets which, in some cases, are large enough to surround a slug. The developmental kinetics of accumulation of this material are those reason- ably expected for a stable sheath component (Fig. 2). No US-insoluble material can be isolated from vegetative cells, from cells developed for up to 8 hr, or from a mutant strain which fails to aggregate. The amount of isolatable material increases between 8 and 12 hr of development, the time when visible sheath first surrounds aggregates. The amount then remains fairly constant for the next 5 hr or so. The decrease in the rate of accumulation of US-insoluble material during this time may occur because a stationary aggregate is already surrounded by the material.

The onset of migration places an increased demand on sheath synthesis. A migrating slug must continually synthe- size new sheath as it migrates, since a trail of spent sheath is left behind. The onset of migration should then result in a greater rate of sheath synthesis and accumulation. Once laid down, the sheath is stable and

will remain intact for many days, so it is unlikely that an increase in synthesis would not be paralleled by an increase in accumulation. The rate of accumulation of the US-insoluble material follows the kinetics expected during migration (Fig. 2).

The US-insoluble material is primarily composed of cellulose. Cellulase-sensitive fibrils were seen by Hohl and Jehli (1973) in surface replicas of trails left behind migrating slugs. They were unable to make similar surface replicas of intact slugs, but electron photomicrographs of the surface sheath show a series of circular profiles whose size and frequency are consistent with them being transverse sections of the fibrils (Farnsworth and Loomis, 1975). Similar profiles have also been seen by George et al. (1972).

The chemical composition of the US-insoluble material and the isolated trails left behind migrating slugs are very similar. Thus, on the basis of size, appearance, developmental kinetics, and chemical composition, we conclude that the US-insoluble material is a component of the surface sheath of D. discoideum.

The US-insoluble sheath of wildtype strain A3 is composed of 60% cellulose, 15% protein, 3% heteropolymer, and 5% lipid. Mutant strains lacking cy-mannosidase, p-glucosidase, and N-acetylglucosaminidase all produce normal amounts of surface sheath whose composition closely resembles that of the sheath from the wildtype cells. Thus, the absence of these enzymes does not seem to affect the composition of the US-insoluble component of the surface sheath.

Two mutant strains lacking UDPG-pyrophosphorylase, which were developed for 18 hr, failed to yield any detectable US- insoluble sheath. These strains are also unable to produce any stalk or spore cells, and they are arrested in development at the onset of culmination (Dimond et al., 1976). They develop morphologically up to this point in a normal fashion, except that the slugs are weak, fragile, and unable to

192 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

migrate. There is an extracellular layer surrounding the aggregates of these strains which is similar in appearance to the nonfibrillar surface sheath described by Farnsworth and Loomis (19751, although it is slightly thinner. These aggregates are devoid of the circular profiles normally seen adjacent to both sides of the nonfibrillar sheath in fixed preparations viewed under the electron microscope (Fig. 4). The failure to isolate US-insoluble sheath from these mutants and the absence of circular profiles from the surface sheath region further suggest that these profiles are transverse sections of cellulose fibrils.

The photomicrographs of transverse sections of normal slugs appear to show a single layer of these circular profiles, which suggests that the US-insoluble fibrillar sheath may be composed of only a single layer of cellulose fibrils. If this is true, the frequency of these flbrils should give an estimate of the amount of cellulose one can expect to find per slug. Farns- worth and Loomis (1975) count about seven circular profiles per micrometer along the sheath, but this number repre- sents only those fibrils having a sufficient electron density to be visible. They estimate that transverse sections of fibrils which are aligned at greater than 15 from the perpendicular of the sections will not be visible. Therefore, the actual frequency of fibrils seen is 30”/180”, or II6 of the total. The density of cellulose is about 1.5 g/cm3, and the total surface area of the slug can be calculated by assuming an average 1 X 0.2-mm slug of lo5 cells. Using these values, we calculate that cellulose should be present at about 280-320 pg/lO’O cells. The data presented here suggest that the amount of cellulose isolated from slugs about to migrate is 360 pg/lO’O cells (Fig. 2). This is in close agreement with the calculated value and suggests that the re- covery of the sheath is good. It further suggests that the surface sheath accounts for all of the US-insoluble material, i.e.,

that the US-insoluble material is only at the surface of the slug and is not in between the cells of the slug. Thus, there seems to be enough cellulose to cover a slug with a single layer of fibrils. These calculations also indicate that the flbrils themselves occupy about 5% of the surface area of a slug.

The fibrillar and nonfibrillar components of the surface sheath appear to be synthesized independently of each other. The nonfibrillar component increases in thickness toward the posterior of the slug as more sheath is added to it (Farnsworth and Loomis, 1975). In contrast, the size and frequency of the circular profiles of the fibrillar component are constant all along the length of the slug. The two sheath components also differ in structure, composition, and function. The US-insoluble component provides a strong, rigid frame- work for the aggregate, which is indispen- sable for migration of the slugs, as shown by the failure of strains Ul and UN1 to migrate. The nonfibrillar, continuous sheath appears to be sufficient to ensure the proper sequence of morphogenetic events up to the migrating slug stage (Di- mond et al., 1976). The chemical nature of this sheath component is unknown, although it probably contains protein.

The surface sheath appears to be ini- tially formed as a viscous liquid which congeals into a rigid casing. The sheath surrounding the tip region of a slug is more distendable than sheath surrounding the posterior (Francis, 1962; Shaffer, 1965; Garrod, 1969) and it appears likely that newly formed sheath, such as that formed early in development, is less rigid than mature sheath. Sheath isolated early in development has less protein, a simpler heteropolymer, and cellulose of a lower crystallinity than sheath isolated late in development (Table 3) (Freeze and Loomis, 1977).

The degree of crystallinity of cellulose profoundly influences its mechanical properties (Hermans, 1949; Howsman and Sis-


son, 1954; Wakeman, 1954; Grant, 1971). High crystallinity is positively correlated with increased strength, rigidity, and hardness. This may help to explain the apparent maturation of sheath during development and along the longitudinal axis of the slug. Table 3 shows a comparison of the relative crystallinities of the sheath isolated from the wildtype at various times in development and the sheath of mutants isolated from migrating slugs. The cellulose from mutant strains lacking a-mannosidase and /3-glucosidase has slightly lower crystallinities than the cellulose from wildtype slugs; slugs of these strains migrate normally. The cellulose isolated from two strains which lack NAG has considerably lower crystallinity values, which are even below those obtained from sheath isolated from the tipless aggregate stage of the wildtype. This is especially interesting in view of the inability of these two strains to migrate as normal-sized slugs. Aggre- gates of these strains often form normal- sized slugs, but they invariably fragment into slugs containing only one-tenth of the number of cells which then migrate at a low rate consistent with their size (Di- mond et al., 1973). This may indicate that the strength of the cellulose fibrils is re- duced in these strains and that they are only able to accommodate the migration of the smaller slug.

The exact mechanism of cellulose biosynthesis and deposition and those factors which determine crystallinity are unknown (Shafizadeh and McGinnis, 19711, but these data suggest that there is a physiologically important maturation of the cellulose of the surface sheath.

This work was supported by NSF Grant GB 29859. We would like to thank Dr. Paul Farnsworth for the electron micrographs and Dr. Stuart Brody for the use of his gas-liquid chromatograph.

REFERENCES

CORBETT, W. (1963). Determination of alpha-cellulose content of cotton and wood cellulose. In “Methods in Carbohydrate Chemistry” (R. L.

Whistler and M. L. Wolfrom, eds.), Vol. 3, pp. 27- 28. Academic Press, New York.

DIMOND, R., BRENNER, M., and LOOMIS, W. (19731. Mutations affecting N-acetylglucosaminidase in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA 70, 3356-3360.

DIMOND, R., FARNSWORTH, P., and LOOMIS, W. (1976). Isolation and characterization of mutations affecting UDPG-pyrophosphorylase activity in Dictyostelium discoideum. Develop. Biol. 50, 169-181.

DIMOND, R., and LOOMIS, W. (1976). Structure and function of P-glucosidases in Dictyostelium discoideum. J. Biol. Chem. 251, 2680-2687.

EVERY, D., and ASHWORTH, J. (1973). The purifica- tion and properties of extracellular glycosidases of the slime mold Dictyostelium discoideum. Bio- them. J. 133, 37-47.

FARNSWORTH, P., and LOOMIS, W. (1975). A gradient in the thickness of the surface sheath of pseudoplasmodia of Dictyostelium discoideum. Develop. Biol. 46, 349-357.

FRANCIS, D. W. (1962). Movement of Pseudoplasmo- dia of Dictyostelium discoideum. Ph.D. thesis, University of Wisconsin, Madison.

FREE, S., and LOOMIS, W. (19751. Isolation of mutations in Dictyostelium disco&urn affecting a- mannosidase. Biochimie 56, 1525-1528.

FREEZE, H., and LOOMIS, W. (1977). Isolation and characterization of a component of the surface sheath of Dictyostelium discoideum. J. Biol. Chem., in press.

GARROD, D. R. (1969). The cellular basis of movement of the migrating grex of the slime mold Dictyostelium discoideum. J. Cell Sci. 4, 781-798.

GEORGE, R. P., HOHL, H. R., and RAPER, K. B. (1972). The ultrastructural development of stalk- producing cells in Dictyostelium discoideum, a cellular slime mold. J. Gen. Microb. 70, 477-489.

GERISCH, G. (1960). Zellfunktion und Zellfunktion- swechsel in der Entwicklung von Dictyostelium discoideum. I. Zellagglutination und Induktion der Fruchtkorperpolaritat. Whilhelm Roux Arch. Entwick Org. 155, 709-729.

GEZELIUS, K., and RANBY, B. (1957). Morphology and fine structure of the slime mold Dictyostelium discoideum. Exp. Cell Res. 12, 265-289.

GRANT, J. (1971). Mechanical behavior of fibers. In “Cellulose and Cellulose Derivatives” (N. Bikales and L. Segal, eds.), Pt. 4, pp. 603-624. Wiley- Interscience, New York.

HEMMES, D., KOJIMA-BUDDENHAGEN, E., and HOHL, E. (1972). Structural and enzymatic analysis of the spore wall layers OfDictyostelium discoideum. J. Ultrastruct. Res. 41, 406-417.

HERMANS, P. (1949). Mechanical properties. In “Physics and Chemistry of Cellulose Fibers.”

HOHL, H., and JEHLI, J. (1973). The presence of cellulose microlibrils in proteinaceous slime track

194 DEVELOPMENTAL BIOLOGY VOLUME 56. 1977

ofDictyostelium discoideum. Arch. Mikrobiol. 92, 179-187.

HOWSMAN, J., and SISSON, W. (1954). Submicros- copic structure. In “Cellulose and Cellulose Deriv- atives” (E. Ott and H. Sprulin, eds.), Pt. 1, pp. 603-624. Wiley-Interscience, New York.

LOOMIS, W. (1972). Role of the surface sheath in the control of morphogenesis in D. discoideum. Nu- ture New Biol. 240, 6-9.

LOOMIS, W. F. (1975). Polarity and pattern in Dic- tyostelium. Mol. Cell Biol. 2, 109-128.

MANN, J. (1963). Fine structure of cellulose. In “Methods in Carbohydrate Chemistry” (R. Whis- tler, and M. L. Wolfrom, eds.), Vol. 3, pp. 108-119. Academic Press, New York.

NICKERSON, R. (1950). The relative crystallinity of cellulose. Aduan. Curb. Chem. 5, 103-125.

NIKAIDO, H., and HASSID. W. (1971). Biosynthesis of saccharides from glycopyranosyl esters of nucleo- side pyrophosphates (sugar nucleotides). Aduan. Curb. Chem. Biochem. 26, 351-483.

RAPER, K. (1935). Dictyostelium discoideum, a new species of slime mold from decaying forest leaves. J. Agr. Res. 50, 135-147.

RAPER, K., and FENNEL, D. (1952). Stalk formation inDictyostelium. Bull. Torrey Bot. Club 79, 25-51.

ROSENESS, P., and WRIGHT, B. (1974). In uiuo

changes in cellulose trehalose and glycogen during differentiation of Dictyostelium discoideum. Arch. Biochem. Biophys. 164, 60-72.

SHAFFER, B. M. (1962). The Acrasina. Aduan. Mor- phol. 2, 109-182.

SHAFFER, B. M. (1965). Cell movement within aggregates of the slime mold Dictyostelium discoideum revealed by surface markers. J. Embryol. Exp. Morphol. 13, 97-117.

SHAFIZADEH, F., and MCGINNIS, G. (1971). Morphol- ogy and biogenesis of cellulose and plant cell walls. Advan. Carb. Chem. Biochem. 26,297-335.

TAKEUCHI, I., and YABUNO, K. (1970). Disaggrega- tion of slime mold pseudoplasmodia using EDTA and various proteolytic enzymes. Exp. Cell Res. 61, 1833190.

TRIPP, V. (1971). Measurement of crystallinity. In “Cellulose and Cellulose Derivatives (N. Bikales and L. Segal, eds.), Pt. 4, pp. 304-324. Wiley- Interscience, New York.

WAKEMAN, H. (1954). Mechanical properties of cellulose and its derivatives. In “Cellulose and Cellu- lose Derivatives” (E. Ott and H. Spurlin, eds.), Pt. 3, pp. 1247-1355. Wiley-Interscience, New York.

WHITFIELD, F. (1964). The use of proteolytic and other enzymes in the separation of slime mold grex. Ezp. Cell Res. 36, 62-72.

the role of the fibrillar component of the surface sheath in the morphogenesis of dictyostelium...

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