Differentiation and secretion in Volvox

JOURNAL OF ULTRASTRUCTURE RESEARCH 70, 318-335 (1980)

Differentiation and Secretion in Volvox M. DAUWALDER, W. G.

WHALEY, AND

R. C.

STARR

Cell Research Institute and Department of Botany, University of Texas at Austin, Austin, Texas 78712 Received October 29, 1979 Synchronously grown populations of the normal HK10 strain of Volvox carteri were used to establish the ultrastructural characteristics of the cells during a maximal phase of sheath secretion. These include evidence for the transport of materials from the rough endoplasmic reticulum to the Golgi apparatus, hypertrophy of the Golgi apparatus, and assembly within the apparatus of a fibrillar material which is subsequently moved in secretory vesicles to the cell surface. The presence of many small vesicles and multivesicular bodies may indicate there are some additional processes related to membrane assembly or turnover. These features were compared with those of a mutant strain which lacks the ability to secrete sheath materials. In the latter, hypertrophy of the apparatus and formation of fibrillar materials was not observed; however, increases in the number of Golgi stacks and various ultrastructural characteristics indicate that membrane assembly continues in the absence of the normal secretory function.

The secretion of cell surface and intercellular matrix components is essential for the integrity and functioning of multicellular organisms. Matrix materials are important not only in providing structural stability to the organism but also in creating the normal milieu in which cells function; and, therefore, these materials are important factors in regulating cellular metabolism and behavior. In addition, they appear to be critical for the normal progress of cellular development and differentiation (see Moscona, 1974; Slavkin and Greulich, 1975; Barondes, 1977). Most surface materials are combinations of protein and carbohydrate. Their composition and preliminary structuring are controlled by the genetic makeup of the cell and change with stages of development. In a wide variety of cells the formation and secretion of intercellular materials has been shown to follow a general pathway with synthesis and initial glycosylation of some protein components in the rough endoplasmic reticulum and synthesis of carbohydrate components and structural remodeling in the Golgi apparatus (see Schachter, 1978; Whaley and Dauwalder, 1979). Once outside the cell, many of the reactions mediated by these constituents can be highly

selective (Dauwalder et al., 1972; Denburg, 1978) implying that a system of informational signals is built into the materials prior to secretion from the cell. Although a great deal of emphasis has been placed on the specificities of proteins and their encoding from DNA, it is becoming increasingly apparent that specificities of carbohydrates may be particularly important in reactions at or outside the cell surface (Hughes, 1975; Marchesi et al., 1978). Since the synthesis of external polysaccharides and the addition of more terminal carbohydrate moieties to other glycosylated materials destined for secretion occurs principally in the Golgi apparatus, the control of saccharide structuring appears to be brought about by processes within this organelle. Such data have led us to interpret the Golgi apparatus as an important site of genetic expression at the post-translational level and to consider the possibility that such control might result from the activities of glycosylating enzymes in combination with the differentiated membranes of the organelle (Whaley and Dauwalder, 1979). Although the general hypothesis of genetic control via the Golgi apparatus is difficult to test directly, one possible approach is in the study of mutants with defective matrix production. 318

0022-5320/80/030318-18502.00/0 Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

DIFFERENTIATION AND SECRETION IN VOLVOX A n u m b e r of such m u t a n t s are available in Volvox, a system which offers a n u m b e r of other advantages for experimentation. Volvox is a simple organism with two distinctive cell types. T h e m a t u r e spheroid consists of somatic cells and gonidial cells which differ in their topographical arrangement, morphology, function, and developm e n t a l potential. T h e spheroid can be studied as an individual, or spheroids can be grown easily in large quantities u n d e r defined conditions of culture to provide materials for biochemical studies. T h e generation time is short, and recently, m e t h o d s have been devised for obtaining populations which develop synchronously. These features allow investigation of biological processes ranging from those concerned with the in vivo interactions in intact organisms to the analysis of reactions at the molecular level. Characteristics of Volvox t h a t have m a d e it an excellent model syst e m for the s t u d y of e m b r y o g e n y and cellular development, the control of sexual differentiation and fertilization, and the genetic analysis of these processes have been previously discussed (Starr, 1970; Kochert, 1975; Wiese, 1976). Since Volvox is a haploid organism, m u t a n t s affecting mating and developmental processes can be isolated readily and then p r o p a g a t e d by asexual reproduction. A n u m b e r of morphogenetic m u t a n t s have been characterized which exhibit discrete effects at specific stages in e m b r y o g e n y (Sessoms and Huskey, 1973). A m o n g these are a group of m u t a n t s which display altered secretory activities. In Volvox carteri, following inversion of the embryo, s h e a t h production is initiated, and the intercellular connections are broken. During subsequent development, the cells of the spheroid are held together by a matrix, or sheath, composed of a b o u t one-third protein and two-thirds polysaccharide (Kochert, 1975; see also Miller et al., 1974). T h e m u t a n t chosen for our initial studies lacks the ability to secrete s h e a t h materials, and after inversion, the spheroid dissociates. Since the m u t a t i o n is

319

t h o u g h t to affect a single genetic locus, its effects are expressed at a particular stage in development, and it clearly involves a m a j o r alteration in secretory activity, this strain seemed to provide a suitable model system for studying the interrelation of the genome, the Golgi apparatus, and developm e n t a l control. T h e preliminary studies reported here compare the cell structure of the n o r m a l spheroid during s h e a t h secretion with those of the mutant. Of particular interest are the morphological differences in the Golgi apparatus. MATERIALS AND METHODS

Volvox carteri f. nagariensis (HK 10 strain) was maintained in culture as previously described (Start, 1969). To produce populations of spheroids which undergo synchronous cycles of asexual reproduction, the cultures are grown in aerated Volvox medium, pH 8 (350 ml medium/500 ml flask), at 28-29°C with 31hr illumination (cool-whitefluorescent tubes, 12 000 lx at growth bench) followedby 17 hr in the dark. Under these conditions one generation is produced every 48 hr, and synchrony can be continued by transfer to fresh media at the beginning of each light period. Cell division and embryo inversion are completed and spheroid enlargement is initiated prior to the release of the young spheroids. When released, the spheroids are enlarging rapidly, and samples were taken at varying times during maximal enlargement. The mutant strain (Start 72-52, Dissociator) was maintained in test tubes, but for experimentation, samples were grown in deep (22 mm) petri dishes to provide a flat surface on which the cells would rest. As far as is known, asexual development proceeds normally through embryo inversion. Following inversion, without either intercellular connections or sheath materials, the cells separate and are handled as a cell culture. For electron microscopy, several variations were tried in the fixation and embedding schedules. Generally good results were obtained with either of two fixation procedures: (a) combined glutaraldehyde-osmium fixation (2% glutaraldehyde:1% osmium tetroxide made up in the normal growth medium containing 1% added sucrose; ff necessary pH was readjusted to 7.7-8.0 with NaOH) for 45 min at 0°C, or (b) glutaraldehyde (3% made up in growth medium, pH 7.7-8.0) for 3 hr at room temperature (if necessary, samples were stored for longer periods in a refrigerator) followed by postfixation in 2% osmium tetroxide (0.01 M cacodylate buffer, pH 7.5-7.7) for 1 hr at room temperature. The buffer systems tested included phosphate, cacodylate, and PIPES (Salema and Brandao, 1973), all of which appeared to give less adequate

320

DAUWALDER, WHALEY, AND STARR

preservation or stabilization of the samples. After fixation, part of the samples were mordanted in tannic acid as suggested by Simionescu and Simionescu (1976). Tannic acid (Baker or Mallinckrodt) was made up in buffer (1% tannic acid, 0.05 M cacodylate, pH 7.3-7.8) or culture medium (1.5% tannic acid, medium adjusted to pH 7.5-7.8). Treatment was for 30 min at room temperature followed by a 5-rain wash in 1% sodium sulfate in 0.05 M cacodylate buffer, pH 7.0. Samples were dehydrated in ethanol or acetone and embedded in Epon-Araldite (Mollenhauer, 1964) or Spurr's (Spurr, 1969) plastics. Samples were agitated during processing and centrifuged between each step. Some samples were put in agar after fixation and handled in agar blocks during subsequent processing. Zeiss equipment was used for light microscopy, and either an RCA 3G or Siemens Elmiskop IA was used for electron microscopy. Preliminary studies indicated that there were possible differences in preservation between the normal spheroids and the mutant cells. It seemed likely that the spheroid matrix, which varies in thickness during this active growth period, might be a factor; and protease digestion (Yates and Kochert, 1976) was used to disperse the cells in an attempt to make all processing steps for the normal cells more comparable to those for the mutant. Protease {Sigma, Type IV) was prepared (1 mg/ml, aqueous) and allowed to stand 30 min at room temperature. An amount was added to cultures to give 100 ~g/ml final concentration, and samples were treated at room temperature for 30 or more min. Since the 30-min treatment gave variable results in different experimental runs, the extent of matrix dissolution was judged visually prior to fixation. As monitored by light microscopy, the protease-treated cells appeared healthy, and somatic cells retained motility even with fairly prolonged treatment; however, preservation of morphology for electron microscopy by the approaches used above was not consistent. It was not possible to determine if protease had damaged

some of the cells or if the treatment had made them somewhat more susceptible to fixation artifact. The results of these experiments will not be reported here.

RESULTS

Effects of Fixation and Processing A number of the common fixation procedures tested 'gave adequate but inconsistent results. In a given sample, some of the cells exhibited blistering or vesiculation of the plasma membrane and varying degrees of swelling associated with the nuclear envelope, rough endoplasmic reticulum, and/ or Golgi apparatus. Mitochondria and chloroplasts were seldom similarly affected. Improved preservation was obtained using glutaraldehyde made up in culture medium; but, for the normal spheroids, the simultaneous glutaraldehyde:osmium fixative (modified from Trump and Bulger, 1966) consistently gave the best preservation of most cellular structures. The plasma membrane was relatively smooth, organelle swelling was minimal, and the "whorling" of membrane structures commonly encountered with primary aldehyde fixation was rare. In gonidial cells, however, profiles of the rough endoplasmic reticulum appeared to be somewhat shorter than those observed with the sequential fixation method, and secretory vesicles were sometimes irregular in contour. Unfortunately, with the

All figures shown except Fig. 10 are of materials which have been mordanted with tannic acid. Fro. 1. (a) Phase contrast micrograph of part of a normal spheroid about 24 hr after release from a synchronous culture. The general cellular characteristics of the gonidial and somatic cells are shown. By electron microscopy these cultures resemble those from unsynchronized samples. Some of the Golgi stacks are indicated by arrows. The gonidial cell is approximately 60 ~m in diameter. Nucleolus of the gonidial cell (NU); condensed material associated with the nuclear envelope in a somatic cell (CNM). (b) Part of a normal spheroid taken from an unsynchronized culture. The cisternae of the Golgi stacks in the somatic cell are not hypertrophied. There is some swelling of the rough endoplasmic reticulum in the region of the Golgi stacks. Glutaraldehyde fixation: osmium postfixation. Pyrenoid (P); dense body (DB). (a), × 1300; (b), × 10 000. FIGS. 2-5. Normal spheroids just released from a synchronous culture and fixed with the simultaneous glutaraldehyde-osmium technique. FIG. 2. A soi~latic cell typical of the active phase of spheroid enlargement. The Golgi cisternae are hypertrophied and as a result the Golgi stacks are expanded. The rough endoplasmic reticulum has a flocculent content and is forming blebs in the region associated with the Golgi stacks. Some small vesicles (single arrows) are associated with the forming secretory vesicles. Rarely, structures resembling "coated pits" are seen along the plasma membrane (double arrows). Nucleus (N); multivesicular body (MVB); vacuoles (V); sheath (S). x 24 000.

X~

O"

322

DAUWALDER,

WHALEY,

mutant strain, the simultaneous fixation appeared too harsh, and membrane breakage was observed. For these samples, the sequential fixation often gave good preservation with smooth plasma membranes and limited organelle swelling. In the n o r m a l spheroids, some shrinkage of the s h e a t h occurs during dehydration. As m o n i t o r e d b y light microscopy, changes in the d e h y d r a t i o n p r o c e d u r e s or use of agar blocks did not p r e v e n t alterations in the sheath. As noted, efforts to c o m p a r e m o r e directly the effects of sample processing on the two strains b y using protease to disperse the n o r m a l spheroids h a v e not yet b e e n successful.

Morphology of the Normal Spheroids I n Volvox each individual, or spheroid, is m a d e up of a p p r o x i m a t e l y 3000 small, flagellated somatic cells located around the p e r i p h e r y with 14-16 large, reproductive gonidial cells which project internally (Fig. 1). In unsynchronized population, cellular m o r p h o l o g y was generally similar to t h a t r e p o r t e d previously (Lang, 1963; K o c h e r t a n d Olson, 1970; P i c k e t t - H e a p s , 1970). Vesicular structures were found in the Golgi region (Fig. 1), b u t the variation f r o m cell to cell m a d e i n t e r p r e t a t i o n of secretory activity difficult (see also, P i c k e t t - H e a p s , 1970). T h i s p r o b l e m was resolved b y studying s y n c h r o n o u s populations just after y o u n g spheroids h a d b e e n released f r o m the parents. T h e rapid e n l a r g e m e n t of these spheroids is primarily due to s h e a t h secretion, and b o t h the somatic and gonidial cells clearly show u l t r a s t r u c t u r a l character-

AND

STARR

istics commonly associated with secretory activity (Figs. 2-5). The rough endoplasmic reticulum contains a flocculent material in the intralumenal space, and blebs are seen in that region located near the forming face of the Golgi apparatus. The Golgi stacks are hypertrophied with distension of the cisternal periphery and formation of secretory vesicles toward the mature face. The hypertrophy seems to be somewhat greater in the somatic cells than that in the gonidia. Fibrillar material can be seen within the Golgi cisternae (Figs. 3 and 5) and in secretory vesicles both associated with the stack and near the plasma membrane (Fig. 3). Some small vesicles appear to be fusing with, or coming from, the more mature Golgi cisternae and the nearly mature secretion vesicles. Numbers of other small vesicles, some of which are coated, are common in the Golgi region as are multivesicular bodies. One feature that differs from that generally seen in most cell types is the pattern of ribosomes bound to the endoplasmic retieulum. Although spirals do occur, surface views commonly show the ribosomes arranged in long double rows (Figs. 6 and 7). Two additional structural characteristics of the normal system will be noted here although they do not appear to have any relation to sheath secretion. First, in somatic cells there is a dense structure located at the periphery of the cell away from the flagellar region. Rough endoplasmic reticulum, containing material of a comparable density, is often associated with this body (Fig. 8). Second, small "circlets" were oc-

FIG. 3. Higher magnification of the Golgi region of a somatic cell similar to that in Fig. 2. The arrows indicate the fibrillar material located in the Golgi cisternae and in the secretory vesicles. When the Golgi stack is sectioned perpendicularly (upper left) the material appears as a fine, feltlike arrangement of fibers, but in oblique sections of the Golgi cisteruae and in the secretory vesicles, the material is seen to be in long fibrillar strands. Membranous structures occur in some of the vacuoles (V). The other large vesicular structures are probably part of the vacuolar system. Multivesicular body (MVB). × 34 000. FIG. 4. Part of the perinuclear region of a gonidial cell during the secretory phase. Golgi stacks with hypertrophied cisternae are scattered throughout the region. Nucleus (N). > 19 800. FIG. 5. Higher magnificatioIi of the Golgi stacks in a gonidial cell. Fibrillar material (arrows) is located in the Golgi cisternae, x 44 000.

FIG. 6. Part of a gonidial cell from a just-released spheroid fixed with glutaraldehyde and postfixed with osmium. The morphology of the Golgi stacks is similar to that in Fig. 5, and faint fibrils can be seen in the cisternae. Short profiles of the ribosomal doublet pattern occur (single arrows) as well as spiral ribosomal arrangements (double arrows), x 42 900. FIO. 7. En face section of the rough endoplasmic reticulum showing the long ribosomal doublet pattern, x 47 500. FIG. 8. Dense body located along the plasma membrane of the somatic cells away from the fiagellar end of the cell. A possible association with the rough endoplasmic reticulum is indicated at the arrow. × 38 000. FIo. 9. Part of the nucleus of a somatic cell from a normal spheroid showing the nuclear "circlets" located in the condensed material associated with the nuclear envelope. Except for the presence of the circlets, the nuclear structure appears normal. The inner surface of the nuclear envelope membrane adjacent to the condensed material differs from the other regions of the envelope membrane with an adherent layer of material (arrow) that is sometimes particulate, x 55 000. FIo. 10. Part of the nucleolus of a mutant gonidial cell showing the "circlets." × 42 700. 324

DIFFERENTIATION AND SECRETION IN VOLVOX casionally seen within the nuclei of both normal and mutant samples. Normal somatic cells do not have a typical nucleolus, and there is a region of condensed material associated with the nuclear envelope. The circlets were located only within the condensed material in somatic cells (Fig. 9) or within nucleoli of gonidial cells (Fig. 10). No elongate profiles were observed, and the structures are presumed to be spheroidal.

Morphology of the Mutant The mutant samples consist of a mixture of individual cells which range in size from somatic-type cells about 10 ~m in diameter through a variety of gonidial cells up to about 80 ~m in diameter, and embryos in different stages of development are included (Figs. 11-14). As judged by the shape of the cells, the embryo in Fig. 13 is in the process of inversion (Viamontes and Kirk, 1977). The embryonic stages have not yet been studied in detail, and the somatic cells have some unusual characteristics which will be described below. The general morphology of most of the gonidial cells is indistinguishable from that of the normal samples; but some binucleate cells were observed (Fig. 12), and occasionally the arrangement of cytoplasmic organelles differed. The rough endoplasmic reticulum was normal in structure and distribution (Fig. 15), and continuities were seen with the outer membrane of the nuclear envelope. Both ribosomal spirals and doublet rows were observed. Blebs were commonly found in the region associated with the forming face of the Golgi apparatus (Fig. 16). The morphology of the Golgi stacks was variable both among cells of different sizes or within an individual cell (Fig. 15). Many of the stacks were normal in shape and number of cisternae, but smaller than in the active control samples due to the lack of hypertrophy. These stacks did not appear to be highly differentiated for secretion. In some stacks, swelling of the cisternal periphery was observed, but evidence for the evolution of normal secretory vesi-

325

cles from the mature face was lacking. Vesicular structures of that size in the Golgi region more often resembled a form of multivesicular body, but there is possibly the evolution of small vesicles. A number of stacks were somewhat unusual in morphology. There was an extension of the cisternae at the mature face; most commonly two cisternae were affected. In profile, the cisternae were longer than those in the rest of the stack and usually curled (Fig. 15). The alteration was not a uniform curling, and characteristically the stacks were asymmetric in appearance. Serial sectioning would be needed to determine if some of the seemingly normal stacks were also modified. Particularly in some of the large gonidia, there was the production of small vesicles with dense contents (Fig. 15). The other features of the Golgi region were comparable to the normal samples. Small vesicles, some coated,.and multivesicular bodies were common. In attempts to sample mutant somatic cell populations, a rather unexpected problem developed. Cells were observed with characteristics intermediate between the normal somatic and normal gonidial cells. In the inverting mutant embryos, gonidia are approximately 12-14 ~m in diameter (Fig. 13), and cells smaller than that should represent somatic cells in stages of enlargement after spheroid dissociation. Cells in the range of 8 to 10 ttm (the normal somatic size) often did not show evidence of flagella in serial 1- to 2-t~m sections viewed by phase microscopy, and some of the cells which retained flagella were larger than expected (Figs. 17-18). Some, but not all, of the enlargement was paralleled by an increase in the size of the vacuoles. The condensed material in the nucleus was more conspicuous than normal and less tightly associated with the nuclear envelope (Figs. 1921). By electron microscopy, this structure resembled a more typical nucleolus (Fig. 22). A number of cells were clearly binucleate (Fig. 22). In such cells, microtubules were located in the cytoplasmic interior.

~Z

t~

L~

DIFFERENTIATION AND SECRETION IN V O L V O X

Observation of living samples showed that there was a class of nonmotile, eye-spotcontaining cells which ranged up to about 15 t~m in diameter. These characteristics suggested that the somatic cell population was not physiologically comparable to that in the normal system. Since the morphology of the Golgi apparatus region fell within the range observed in the gonidial cells, the latter were primarily used for analysis of the organeUe. In none of the experiments with the mutant was there evidence of sheath material at the cell surface. During the progress of the experiments, a large number of Golgi stacks was noted in some of the mutant cells, and the question arose as to whether there were differences in stack number from those in the normal cells. The contrast of the samples with tannic acid mordanting allows relatively consistent counting of Golgi stacks in l-t~m thick sections using phase contrast microscopy (see Figs. 1, 11, 12, and 14). Counting and measurements were done on sections of cells which included nuclei. The data are summarized in Table I. The mutant cells showed an increasing number of Golgi stacks with increasing cell size. In the smallest diameter range, obvious somatic-type cells were excluded, but due to the uncertainties discussed above, some somatic cells may have been included in the counting data. In the normal spheroids, although the size of the gonidia varies from about 45 to

327

60 ~m, those in each experiment are fairly uniform in diameter. Samples were chosen which had gonidia at either end of the normal range, and matching samples were selected from the mutant data. The differences between the normal and mutant cells are probably within the experimental error. Although more than 4000 Golgi stacks have been scored, the numbers are considered to be approximations. A more quantitative study was not undertaken primarily because the visibility of the Golgi region is affected by vacuolar preservation, and it is difficult to insure comparability of vacuolar shape among the experimental runs. The data clearly show an increase in the number of Golgi stacks with gonidial enlargement, and make it unlikely that there are large differences in the number between normal and mutant cells. Somatic cells averaged about five stacks per section in all samples counted. The increase in the number of Golgi stacks is perhaps more striking when considered on a per cell basis. For example, counts were made from photographs of 10 serial sections of the gonidial cell shown in Fig. 14. Counts from photographs are lower than those obtained with microscopic observation, but it is less likely that one stack will be scored on two adjacent sections (the stacks are in the 1-~m size range). The total number of stacks was 234. Since the first section counted included the periphery of

FIGS. 1I AND 12. Phase contrast micrographs of cells of the mutant strain. Figure 11 shows the typical structure of the mutant cells and illustrates the variability in cell size. Figure 12 shows an example of a binucleate gonidial cell and of a cell with a somewhat uncharacteristic vacuolar arrangement. × 920. FIG. 13. Phase contrast micrograph of an inverting embryo from the mutant strain. The gonidial cell is approximately 12 ~m in diameter. × 1140. FIG. 14. Phase contrast micrograph of a mutant gonidial cell approximately the same size as the normal gonidial cell in Fig. la. The cell structure is indistinguishable from that of the normal. The gonidial cell is about 58 tLm in diameter; and in the counting data, this section was scored as having 25 Golgi stacks, x 1140, FIG. 15. Perinuclear region from a large gonidial cell of the mutant. This one region illustrates the different forms of the Golgi stacks found in the mutant cells of varying sizes. Some stacks (No. 1) appear relatively inactive, some (No. 2) exhibit peripheral swelling of the cisternae, some (No. 3) show the elongated and curled cisternae at the mature face, and some (No. 4) are involved in the elaboration of a small, dense vesicle found in a few of the cells. The cell was over 70 ~m in diameter. Glutaraldehyde fixation:osmium postfixation, x 10 000. FIG. 16. Higher magnification of the Golgi stacks from a smaller (36 ~m) gonidial cell of the mutant strain illustrating the continued blebbing of the rough endoplasmic reticulum, and some curling of the Golgi stacks. x 6 4 000.

328

DAUWALDER, WHALEY, AND STARR

FIGs. 17-21. These figures were all printed together to ensure equality of magnification. FIGs. 17 AND 18. Phase contrast micrographs of serial sections of a somatic cell from the mutant strain. The presence of flagella indicate that the cell is of the somatic type, but both the cell (about 11 #m) and the vacuoles within the cell are larger than those from a normal spheroid (compare Fig. 21). × [720 Fins. 19 AND 20. Phase contrast micrographs of somatic cells in the mutant strain which closely resemble the normal somatic cells in size (8-9/~m) and structure except that the condensed material in the nucleus (arrows) is enlarged and less closely associated with the nuclear envelope. × 1720. FzG. 21. Phase contrast micrograph showing several somatic cells from the normal spheroid for comparison with Figs. 17-20. Cross-sections of the cells are shown with the spheroid periphery to the right. The normal configuration of the condensed nuclear material is indicated by arrows. Somatic cells sectioned longitudinally are shown in Fig. la. Dense body (DB); pyrenoid (P). > 1720. FIG. 22. Part of a somatic cell from the mutant strain similar to that in Fig. 20 showing the changes in the condensed nuclear material. This cell is binucleate (N). The Golgi cisternae are not hypertrophied, and the stack is about the same size as those in the mutant gonidial cell shown in Fig. 16. There is some fixation damage.

x 16 000. the nucleus, and the last the periphery of p o r t u n i t y to s t u d y f a c t o r s i n v o l v e d in t h e the nucleolus, this probably represents c o n t r o l o f d i f f e r e n t i a t i o n a n d d e v e l o p m e n t , more than half, but less than two thirds of a n d t o r e l a t e c h a n g e s i n d e v e l o p m e n t a l p a t the total number in the cell. Figure 14 was t e r n s to a l t e r a t i o n s a t t h e c e l l u l a r level. A g r e a t d e a l of w o r k h a s p o i n t e d to t h e cell taken from section 8 in the series. DISCUSSION The availability of a series of morphogenetic mutants in Volvox offers a rare op~

s u r f a c e as a n i m p o r t a n t site in t h e m e d i a t i o n o f d e v e l o p m e n t a l p r o c e s s e s , a n d differe n c e s in s u r f a c e r e s p o n s e s a r e o f t e n a n int e g r a l p a r t o f t h e p r o g r e s s i v e c h a n g e s in

DIFFERENTIATION

329

A N D S E C R E T I O N IN V O L V O X TABLE I

CHANGES IN THE GOLGI STACKS WITH CELL SIZE C o m p a r i s o n of t h e n o r m a l a n d m u t a n t gonidia (diam range)

M u t a n t CeUs Diameter range a U p to 25 26-45 46-65 Above 65

Normal

Golgi stacks b 13 22 30 43

Average d i a m e t e r Golgistacks No. ofceHs c o u n t e d

Mutant

40-50

55-65

40-50

55-65

48 21 22

63 43 22

45 26 23

59 33 24

a D i a m e t e r is expressed in m i c r o m e t e r s . b Golgi stacks are given as t h e average n u m b e r / c e l l / s e c t i o n .

cellular function. Studies attempting to more clearly define the nature of the interactions involved are largely being pursued in animal systems, but the characteristics of the mutant strain studied here would seem to make it particularly useful for assessing the importance of surface activities in a plant system. Gonidial development and early embryogenesis appear to proceed normally in the absence of sheath material, and the lack of this material should make it possible to examine the surface features of these stages by a variety of techniques that are difficult to use with most plant cells. Moreover, our initial studies of this strain indicate that it may also be possible to follow the assembly and translocation of surface membrane in these cells as a process separate from the secretion of sheath constituents. Before proceeding to a more detailed consideration of these findings, since ultrastructural studies of Volvox are somewhat limited, some general comments on the system seem warranted. To begin with, the use of varying procedures in comparing the characteristics of the normal and mutant cells is not entirely satisfying. Differences in the response of tissues and individual cells to fixation are, however, common; and the inherent structural dissimilarities of the normal ensheathed spheroid and the mutant cells undoubtedly contributed to the problems of achieving uniform results. In addition, some controversy has surrounded the use of mixtures of glutaraldehyde and

osmium as fixatives (see Hopwood, 1972). Recent studies illustrate the contrasting results that have been obtained. Hasty and Hay (1978) presented evidence that simultaneous glutaraldehyde-osmium fixation improves the preservation of the plasma membrane and eliminates many of the artifacts that may result with primary aldehyde fixation. On the other hand, Kalderon and Gilula (1979) discussed the possibility that this method disrupts membrane structure and produces "lesions" in the cell. Somewhat strangely, our findings are compatible with both these studies. Preservation of the somatic cells in the normal spheroid was much improved, whereas in t h e mutant, structures similar to those described by Kalderon and Gilula (1979)were observed with evidence of membrane breakage or of membrane regions in which only half the membrane appeared to have been preserved. This problem with the mutant strain will be pursued further, since it has been reported that simultaneous glutaraldehyde-osmium fixation followed by postosmication (Franke et al., 1969) improves the retention of the structure of algal surface glycoproteins (Roberts, 1974). Although the nature of the fibrillar component of the Volvox sheath has not been established, both intra- and extracellular fibrils are generally more apparent with the simultaneous fixation. Modifications in this method may help clarify the situation in the mutant strain. Viruses and virus-like particles have now

330

DAUWALDER, WHALEY, AND STARR

been reported in many classes of algae (see for review, Sherman and Brown, 1978). Most of these differ in morphology and/or location from the intranuclear spheroids described here, but some of the structures are similar. For example, in one of the cases reported by Mattox et al. (1972) aggregations of spherical inclusions were noted in heterochromatic regions of the nucleus; and in a viral infection studied by Lee (1971) although the particles were predominantly cytoplasmic, a few were located in the nucleolus. Quite similar structures have also been found in cultured plant cells (Sjolund and Shih, 1970; Endress and Sjolund, 1976), and their presence is correlated with a loss of ability of the cultured cells to differentiate and with alterations in cellular morphology. Whether these structures are viral in character is still unclear. In our studies there was no evidence for virus production, nor was the presence of the particles correlated with alterations in cellular morphology or growth of the normal spheroids. Since some of the characteristics of the mutant strain are reminiscent of effects seen in virally infected algae (abnormal differentiation, lack of wall synthesis, and presence of naked stages that might facilitate transmission; see Toth and Wilce, 1972), cells of the mutant were carefully checked for any evidence that viral effects contributed to the differences in morphology. None was observed, and the growth and reproduction of the mutant and lack of cellular lysis make it unlikely that virus elaboration is involved. Identical structures have been observed in strains of Volvox carrying the noninducibility locus (Jeffrey Zeikus, personal communication), and although they were more common in occurrence than in our study, no other effects were evident. Further studies will be needed to determine if these structures represent some latent form of virus, and until then their presence in algal systems should be viewed with caution. The presence of intravacuolar structures or multivesicular bodies is often indicative

of lysosomal activity. Such activities are in general much less well defined in plant systems than those in animals (Matile, 1975, 1978; Holtzman, 1976). Commonly, the plant cell vacuole has been implicated as the site of lysosomal degradation. Previous studies of algae and other plant cells have shown that in some cases the occurrence of intravacuolar structures and indications of acid phosphatase activity are both increased in relation to aging (Palisano and Walne, 1972; Gomez et al., 1974a,b; Matile, 1975). Although the somatic cells in this system are postmitotic and will eventually undergo senescence after the next reproductive cycle, both intravacuolar membranes and multivesicular bodies are prevalent in cells of the just-released spheroids suggesting autophagic processes may be functionally important prior to the onset of senescence. The multivesicular bodies seen here are very similar in morphology to those commonly observed in animal cells. In the latter, they are thought to participate in membrane turnover either from the Golgi apparatus or the plasma membrane or in autophagic activities (Holtzman, 1976). In this system, as in many other secretory plant cells, the morphology is consistent with the transport of a large amount of membrane from the Golgi apparatus to the plasma membrane (for estimates of the rate and amount of membrane transported, see Brown, 1969; Ramus and Robins, 1975; Schnepf and Busch, 1976); however, in plant cells there is little evidence for the return of membrane from the plasma membrane comparable to that characteristic of many animal cells (Silverstein et al., 1977). In cells that are not rapidly increasing in size, as the somatic cells here, some mechanism for balancing membrane addition must be operative. The question of the degradation or reutilization of membrane components as a consequence of secretory activity remains a sticky one in plant cells, in part, because of the difficulties in using various tagging techniques for studying endocytic activities. The mutant described

DIFFERENTIATION AND SECRETION IN VOLVOX here may provide a system in which this possibility can be tested. Keys to the control of development have long been sought, and recent evidence suggests that factors which control the synthesis and transport of cell surface constituents may be important determinants of cellular differentiation. This laboratory has been particularly interested in the potential informational qualities of the carbohydrate moieties of surface macromolecules and in trying to define more clearly the processes in the Golgi apparatus that may be involved in regulating the assembly of these substances. Previous studies in algal systems have shown that normal morphogenesis is in some cases related to the characteristics of surface polysaccharides (Bryhni, 1974; Hogsett and Quatrano, 1978) and that glycoproteins play a role in surface interactions, particularly in fertilization and in the induction of sexuality (Wiese, 1974; Kochert, 1977; Callow et al., 1978b). The assembly of algal polysaccharides has been most extensively studied in the red and brown algae, and the evidence indicates that, as in other systems, polysaccharides are synthesized and sulfated in the Golgi apparatus (Coughlan and Evans, 1978; Callow et al., 1978a; McCandless and Craigie, 1979). Study of plant glycoproteins is more recent than that of the animal glycoproteins and much less is known about the pattern of assembly {Sharon, 1974; Chrispeels, 1976), but similarities with the general scheme established for animal cells (Schachter, 1978) seem likely. The ultrastructure of the cells of the justreleased normal spheroids is consistent with the assembly scheme proposed for the production of protein-polysaccharides, with sequential involvement of the rough endoplasmic reticulum and the Golgi apparatus. The significance of the differences in ribosomal patterning is not known, but the doublet strands were noted in some of the earliest electron microscope studies of Volvox (Lang, 1963). They are seen only associated with the endoplasmic reticulum or

331

nuclear envelope and do not appear to be similar to aggregates of ribosomes induced by certain treatments {Maraldi et al., 1973; Dondi and Barker, 1974; Kusamrarn et al., 1975) or to arrangements reported in some virally infected algal cells (Hoffman and Stanker, 1976). Whether there are changes in the ribosomal patterns with development and maturation has not been studied, but the doublet strands are readily apparent in unsynchronized cultures, in synchronized cultures past the peak of secretory activity, and in the mutant cultures. Evidence has been presented for specificity in the interactions between the membranes of the endoplasmic reticulum and the ribosomes, and theories as to the generation of ribosomal patterns have been discussed (Sabatini and Kreibich, 1976). Here, two distinctive patterns coexist, and questions as to the control of the associations involved and their functional relevance pose intriguing problems for research. The interrelation between the rough endoplasmic reticulum and the Golgi apparatus is morphologically more similar to that commonly seen in animal systems than it is to that of higher plants. Within this region there are obvious blebs from the endoplasmic reticulum near the forming face of the Golgi apparatus, numbers of small vesicles, and multivesicular bodies. In higher plants, even in cells in which masses of materials are being rapidly moved in membranebounded vesicles from the Golgi apparatus to the cell surface, endoplasmic reticulum blebbing is not usually evident. The vesicles from the endoplasmic reticulum are thought to act in the transport of the secretory materials for further assembly in the Golgi apparatus. They are often numerous in cells where masses of protein or incompletely glycosylated proteins are moved relatively rapidly from the rough endoplasmic reticulum to the Golgi apparatus, as would be consistent with cellular function in the normal spheroids. However, endoplasmic reticulum blebbing seems to be equally common in the mutant cells, and numbers

332

DAUWALDER, WHALEY, AND STARR

of small vesicles are observed in the Golgi region. This could indicate that there is some transport of secretory materials from the endoplasmic reticulum to the Golgi apparatus in the mutant cells, or alternately, that the vesiculation of the rough endoplasmic reticulum is more closely related to the pattern of membrane assembly in the cells than has previously been assumed. In the Golgi apparatus of the normal spheroid cells, most of the polysaccharides of the sheath are probably synthesized and the glycosylation of sheath glycoproteins completed. With respect to glycoprotein assembly the elaboration of the rough endoplasmic reticulum, hypertrophy of the Golgi apparatus, and organization of a fibrillar surface component reported here have a number of similarities to the study of Minami and Goodenough (1978) on gametic cell fusion in Chlamydomonas. Using combined biochemical and ultrastructural approaches they showed that fusion of the cells induced the synthesis of novel glycoproteins (glycopolypeptides) and their secretion into the zygote cell wall. Coincident with the onset of synthetic activity fibrillar materials are organized in the Golgi apparatus. In Volvox it remains to be established whether the fibrillar component is glycoprotein in nature, but it is clearly assembled in the Golgi apparatus; either it is synthesized, or as a result of other activities, organized into a fibrillar form (for example, Weinstock and Leblond, 1974). The somatic cells in Volvox have most commonly been considered with regard to secretion, but the gonidia are also secretory, and the materials surrounding the gonidial cells come to almost fill the interior of the spheroid. Whether this material is compositionally the same as the sheath has not been established. Both have a fibrillar component, both are more highly organized near the cell surface, and both are dispersed by protease. Although the possibility that conformational changes occur during dehydration cannot be ruled out, the morphology of the materials is somewhat different, and the composition of the

spheroid surface may be distinctive (Burr and McCracken, 1973). The function of the Golgi apparatus in the mutant strain is difficult to interpret. The samples are by nature heterogeneous and some of the variations in form resemble those reported in normal Volvox populations containing mixed stages of growth. The most consistent difference is the lack of Golgi apparatus hypertrophy and the absence of the large secretory vesicles. This seems to imply disruption in the synthesis and secretion of all of the sheath materials; however, it is possible that some assembly occurs at a lower rate or periodically within the cells. The peripheral swelling of the Golgi apparatus cisternae observed sporadically in the mutant strain could be indicative of such processes, and transport of materials might occur in small vesicles. If transport to the cell surface takes place, the material does not remain associated with the cell in a visualizable form; but many factors can influence the localization of surface materials, as, for example, changes in surface binding, changes in the interactions among the various constituents involved in the stability of the structure, or the release of lytic enzymes. The Golgi stacks with the long, asymmetrically curled profiles at the mature face are somewhat abnormal in appearance. Curling is often a normal feature of Golgi apparatus structure, but it is usually of a more symmetrical nature. Although curling alone is not a reliable indicator of altered function, our previous work with the corn root tip indicated that the use of some metabolic inhibitors which decreased the amount of secretory activity also increased the amount of cisternal curling (see Whaley et al., 1975). In both the treated corn root tips and the Volvox mutant, the assembly of secretory material can be markedly diminished, whereas, the synthesis and assembly of membrane components appears to continue. The evidence for membrane assembly is much more striking in the Volvox mutant, as shown by the normal in-

DIFFERENTIATION AND SECRETION IN VOLVOX crease in the number of Golgi stacks and in cell size. Presumably these membranes contain glycoproteins, and therefore it appears that the control of the assembly of membrane glycoproteins is separable from that of secretory glycoproteins. An inconsistency encountered in this study may actually represent a most interesting phenomenon--the unusual characteristics of some of the somatic cells of the mutant strain. In normal spheroids the somatic cell line is separated from the gonidial line by a particular cell division during embryogenesis. After this division the gonidial cells cease to divide and undergo maturation. They retain the potential for development (as a germ line cell) and are subsequently responsible for the formation of new asexual individuals or the production of eggs and sperm during sexual reproduction. In the embryo after the stage of gonidial cell formation, the somatic cells continue to divide until the mature number of cells is reached. At this point they normally undergo a limited amount of growth; they function in sheath secretion, motility, and photoreception; but they lack the capacity to divide or differentiate further. In the mutant, the somatic cells enlarge, and a number of them are binucleate and show alterations in microtubule location. These features may indicate that the cells have retained some potential for cellular division. Binucleate somatic cells have been reported as an abnormality in Volvox tertius (Pickett-Heaps, 1970), but none was observed in any of the experiments with the normal strain in this study. The changes in nuclear structure would also be consistent with the possibility that the cells have some increased genetic activity as compared to the normal somatic cells. Two of the possible interpretations are that (1) the lack of sheath materials has affected the normal programming of these cells, or (2) the "switching on" of sheath secretion is in some way connected to the "switching oft"' of characters related to the capacity to differentiate.

333

Some observations consistent with the latter suggestion have been made in another mutant of V. carteri f. nagariensis. In this "regeneration" strain some of the somatic cells in the spheroid do not lose the potential for development and can undergo asexual and sexual phases of reproduction. These cells can be readily identified because they are enlarged and because they secrete less sheath material (see Fig. 33, Start, 1970). The number of somatic cells involved varies suggesting that regulatory factors can indeed modify the expression of the normal program leading to terminal differentiation and eventual senescence in some percentage of the cells in the somatic line. In the mutant studied here, there is no evidence that the enlarged somatic cells retain further developmental capacity, and presumably they eventually undergo senescence. A few observations made during the study may be relevant to this process. Bodies which are morphologically definable as microbodies are observed in all cell types. Their activities in algae have not been well defined (for a review of higher plant microbodies, see Beevers, 1979), and in normal algal cultures peroxidase staining may be light or absent (Gomez et al., 1974b; Rogalski et al., 1977). However, in senescent cultures of Euglena, Gomez et al. (1974b) reported an increase in the size of the microbodies and an increase in peroxidase staining. In some cells of the mutant, microbodies were much larger and sometimes more regularly spherical in contour. Whether this change was limited to the small cell class has not yet been determined. The point at which the genetic defect modifies the assembly pathway in the mutant strain is not known. The morphological characteristics could be interpreted as indicating a lack of synthesis of the protein component and hence failure of secretory glycoproteins to develop. If so, the alteration would likely be at some site affecting the transcriptional or translational level of genetic control. Still, the general absence of evidence for the formation of secretory

334

DAUWALDER, WHALEY, AND STARR

products and the alterations of some of the Golgi stacks associated with the presence of the mutation could imply modifications at the post-translational level, perhaps at the level of the Golgi apparatus. Whatever makes for the lack of formation of secretory products, there is apparently not a comparable interruption of the formation of membranes in the organelles of the secretory pathway. The evidence suggests that membrane biogenesis continues and that membrane assembly in the Golgi apparatus and possibly its transport to the cell surface are not basically affected by the mutation. The plasma membranes of the mutant gonidia are normal with regard to the induction of sexual reproductive cycles which indicates that functional receptors for the inducer are present and that membrane interactions involved in the induction process are relatively undisturbed. If one reasons from the fact that most cell membranes have glycosylated components which are important in membrane function, then the Golgi apparatus in the mutant appears capable of carrying out the glycosylation reactions necessary for membrane biogenesis even though the formation of secretory products is curtailed. These conclusions are generally consistent with the idea that a fundamental function of the Golgi apparatus is as a site of assembly and differentiation of membrane (see SjSstrand, 1968; Whaley and Dauwalder, 1979) and that a major part of this function is related to the characterization of membrane constituents destined for the plasma membrane. The latter function appears to be separable from the differentiation of the organelle associated with the production of secretory materials. The authors wish to thank Marie Morgan and Judy Edwards for expert technical assistance. Portions of this study were presented at the 37th Annual Meeting of the Electron Microscopy Society of America, EMSA Proceedings, page 338, 1979, and part of it at the 19th Annual Meeting of the Society for Cell Biology. REFERENCES BARONDES, S. H., Ed. (1977) Neuronal Recognition, Plenum, New York.

BEEVERS, H. (1979) Annu. Rev. P l a n t Physiol. 30, 159-193. BROWN, R. M., JR. (1969) J. Cell Biol. 41, 109-123. BRYHNI, E. (1974) Develop. Biol. 37,273-279. BURR, F. A., AND MCCRACKEN,M. E. (1973} J. Phycol. 9, 345-346. CALLOW, M. E., COUGHLAN,S. J., AND EVANS, L. V. (1978a) J. Cell Sci. 32, 337-356. CALLOW, M. E., EVANS, L. V., BOLWELL, G. P., AND CALLOW, J. A. (1978b) J. Cell Sci. 32, 45-54. CI-IRISPEELS, M. J. (1976) Annu. Rev. Plant Physiol. 27, 19-38. COUG~ILAN, S. J., AND EVANS, L. V. (1978) J. Exp. Bot. 29, 55-68. DAUWALDER, M., WHALEY, W. G., AND KEPHART, J. E. (1972} Sub-Cell. Biochem. 1, 225-275. DENBURG, J. L. (1978) Advan. Comp. PhysioL Biochem. 7, 105-226. DONDI, P. G., AND BARKER, D. C. (1974) J. Cell Sci. 14, 301-317. ENDRESS, A. G., AND SJOLUND, R. D. (1976) Amer. J. Bot. 63, 1213-1224. FRANKE, W. W., KRIEN, S., AND BROWN, R. M., JR. (1969) Histochemie 19, 162-164. GOMEZ, M. P., HARRIS, J. B., AND WALNE, P. L. (1974a) Brit. Phycol. J. 9, 163-174. GOMEZ, M. P., HARRIS, J. B., AND WALNE, P. L. (1974b) Brit. Phycol. J. 9, 175-193. HASTY, D. L., AND HAY, E. D. (1978) J. Cell BioL 78, 756-768. HOFFMAN, L. R., AND STANKER, L. H. (1976) Canad. J. Bot. 54, 2827-2841. HOGSETT, W. E., AND QUATRANO,R. S. (1978) J. Cell BioL 78, 866-873. HOLTZMAN, E. (1976) Lysosomes: A Survey (Cell Biology Monographs, Vol. 3), Springer-Verlag, Vienna. HoPwooD, D. (1972) Histochem. J. 4, 267-303. HUGHES, R. C. (1975) Essays Biochem. 11, 1-36. KALDERON, N., AND GILULA,N. B. (1979) J. Cell Biol. 81, 411-425. KOCHERT, G. (1975) in MARKERT, C. L., AND PAPACONSTANTINOU, J. (Eds.), The Developmental Biology of Reproduction, pp. 55-90, Academic Press, New York. KOCHERT, G. (1977) in O'DAY, D. H., AND HORGEN, P, A. (Eds.), Eucaryotic Microbes as Model Developmental Systems, pp. 235-251, Dekker, New York. KOCHERT, G., AND OLSON, L. W. (1970) Arch. MikrobioL 74, 19-30. KUSAMRARN, W., SOBHON, P., AND BAILEY, G. B. {1975) J. CellBiol. 65, 529-539. LANG, N. J. (1963) Amer. J. Bot. 50, 280-300. LEE, R. E. (1971) J. Cell Sci. 8, 623-631. MARALDI, N. M., BIAGINI, G., SIMONI, P., BARBIERI, M., MARINI, M., ANn BERSANI, F. (1973) J. Ultrastruct. Res. 44, 265-278. MARCHESI, V. T., GINSBURG,V., ROBBINS,P. W., AND Fox, C. F., Eds. (1978) Cell Suface Carbohydrates and Biological Recognition, Alan R. Liss, New York.

DIFFERENTIATION AND SECRETION IN VOLVOX MATILE, P. (1975) The Lytic Compartment of Plant Cells (Cell Biology Monographs, Vol. 1), SpringerVerlag, Vienna. MATILE, P. (1978) Annu. Rev. Plant Physiol. 29, 193213. MATTOX, K. R., STEWART, K. D., AND FLOYD, G. L. (1972) Canad. J. Microbiol. 18, 1620-1621. McCANDLESS, E. L., AND CRAIGIE, J. S. (1979) Annu. Rev. Plant Physiol. 30, 41-53. MILLER, D. H., MELLMAN, I. S., LAMPORT, D. T. A., AND MILLER, M. (1974) J. Cell Biol. 63, 420-429. MINAMI, S. A., AND GOODENOUGH, U. W. (1978) J. Cell Biol. 77,165-181. MOLLENHAUER,H. H. (1964) Stain Technol. 39, 111114. MOSCONA, A. A. ED. (1974) The Cell Surface in Development, Wiley, New York. PALISANO, J. R., AND WALNE, P. L. (1972) J. Phycol. 8, 81-88. PICKETT-HEAPS, J. D. (1970) Planta 90, 174-190. RAMUS, J., AND ROBINS, D. M. (1975) J. Phycol. 11, 70-74. ROBERTS, K. (1974) Phil. Trans. R. Soc. London Ser. B 268, 129-146. ROGALSKI, A. A., OVERTON, J., AND RUDDAT, M. (1977) Protoplasma 91, 93-106. SABATINI, D. D., AND KREIBICH, G. {1976) in MARTONOSI, A. (Ed.), The Enzymes of Biological Membranes, Vol. 2, pp. 531-579, Plenum, New York. SALEMA, R., AND BRANDAO,I. (1973) J. Submicrosc. Cytol. 5, 79-96. SCHACHTER, H. (1978) in HOROWITZ, M. I., AND PIGMAN, W. (Eds.), The Glycoconjugates, Vol. 2, pp. 87-181, Academic Press, New York. SCHNEPF, E., AND BUSCH, J. (1976) Z. Pflanzenphysiol. 69, 62-71. SESSOMS, A. H., AND HUSKEY, R. J. (1973) Proc. Nat. Acad. Sci. USA 70, 1335-1338.

335

SHARON, N. (1974) in PRIDHAM, J. B. (Ed.), Plant Carbohydrate Biochemistry, pp. 235-252, Academic Press, New York. SHERMAN, L. A., AND BROWN, R. M., JR. (1978) in FRAENKEL-CONRAT, n., AND WAGNER, R. R. (Eds.), Comprehensive Virology, Vol. 12, pp. 145234, Plenum, New York. SILVERSTEIN, S. C., STEINMAN, R. M., AND COHN, Z. A. (1977) Annu. Rev. Biochem. 46, 669-722. SIMIONESCU, N., AND SIMIONESCU,M. (1976) J. Cell Biol. 70, 608-621. SJOLUND, R. D., AND SHIH, C. Y. (1970) Proc. Nat. Acad. Sci. USA 66, 25-31. SJ()STRAND, F. S. (1968) in DALTON, A. J., AND HAGUENAU, F. (Eds.), The Membranes, pp. 151-210, Academic Press, New York. SLAVKIN,H. C., AND GREULICH, R. C. (Eds.) {1975) Extracellular Matrix Influences on Gene Expression, Academic Press, New York. SPURR, A. R. (1969) J. Ultrastruct. Res. 26, 31-43. STARR,R. C. {1969) Arch. Protistenk. 111, 204-222. STARR,R. C. (1970) Develop. Biol. Suppl. 4, 59-100. TOTIt, R., AND WILCE, R. T. (1972) J. Phycol. 8, 126130. TRUMP, B. F., AND BULGER, R. E. (1966) Lab. Invest. 15, 368-379. VIAMONTES, G. I., AND KIRK, D. L. (1977) J. Cell Biol. 75, 719-730. WEINSTOCK, M., AND LEBLOND, C. P. (1974) J. Cell Biol. 60, 92-127. WHALEY, W. G., AND DAUWALDER, M. (1979) Int. Rev. Cytol. 58, 199-245. WHALEY,W. G., DAUWALDER,M., ANDLEFFINGWELL, T. P. (1975) Curr. Top. Develop. Biol. 10, 161-186. WIESE, L. (1974) Ann. N.Y. Acad. Sci. 234, 383-395. WIESE, L. (1976) in LEWIN, R. A. (Ed.), The Genetics of Algae, pp. 174-197, Blackwell, Oxford. YATES, I., AND KOCHERT, G. (]976) Cytobios 15, 7-21.