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1973 The trs ucture, arrangement and intracellular origin of tubular mastigonemes in Ochromonas minute and Monas sp (Chrysophyceae) Francis Gordon Hill Iowa State University

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HILL, Francis Gordon, 1947- THE STRUCTURE, ARRANGEMENT AND INTRACELLULAR ORIGIN OF TUBULAR MASTIGONEMES IN OCHRQMONAS MINUTE AND NONAS SP. (CHRYSOPHYCEAETT^

Iowa State University, Ph.D., 1973 Biology

University Microfilms, A XEROX Company, Ann Arbor, Michigan The structure, arrangement and intracellular origin

of tubular mastigonemes in Ochromonas minute and

Monas sp. (Chrysophyceae)

by

Francis Gordon Hill

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of

The Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Major: Cell Biology

Approved:

Signature was redacted for privacy. In Chcfrge of Major Work

Signature was redacted for privacy. Chairman, Advisory Commitfee Cell Biology Program

Signature was redacted for privacy. For the Graduate College

Iowa State University Ames, Iowa

1973 ii

TABLE OF CONTENTS Page INTRODUCTION 1

LITERATURE REVIEW 3

Terminology and Mastigoneme Fine Structure 3

Taxonomic Distribution 5

Differences in Mastigoneme Fine Structure Among Taxa 6 Arrangement of Mastigonemes on the Flagellum 8

Molecular Architecture of Mastigonemes 9

Attachment of Mastigonemes to the Flagella 12

Origin and Assembly of Mastigonemes 13

Release of Mastigonemes to the Flagellum 18

Control of Mastigonemogenesis 20

Mastigoneme Function 20 MATERIALS AND METHODS 24

Culture Conditions 2 4

Deflagellation and Kinetics of Reflagellation and Mastigoneme Transport 25

Light Microscopy 25 Electron Microscopy 26

Sectioned material 26 Whole mount material 27 Microscopy 27 RESULTS 28

General Morphology 2 8 iii

Page Mastigoneme Structure, Arrangement and Attachment 29

Mastigoneme Morphogenesis and Transport to the Flagellum 31

Analysis of the Intracellular Location of Pre­ sumptive Mastigonemes During Flagellar Re­ generation and Colchicine Inhibition 34 DISCUSSION 59

Phyletic Relationship of Monas to 0. minute 59

Arrangement of Mastigonemes Relative to their Function 60

Attachment of the Mastigoneme to the Flagellum 61

Intracellular Development, Transport and Release of Mastigonemes 62

Golgi apparatus function 64 Control of mastigoneme arrangement 6 7 Nuclear envelope-perinuclear space function 6 8

Kinetics of Mastigoneme Assembly and Transport 72

CONCLUSION 79 BIBLIOGRAPHY 80

ACKNOWLEDGMENTS 91 1

INTRODUCTION

Hair-like flagellar appendages (mastigonemes) were first reported by Loeffler (65) in 1889 in the chrysophycean Monas.

Subsequently, Fischer (34) , using the same method, a mordant and staining technique developed for bacterial flagella, described a variety of arrangements of mastigonemes on the flagella of many taxa. The existence of mastigonemes was still in question however because of the harsh technique that was used to visualize them. Deflandre (24, 25) demonstrated mastigonemes in 19 34 using nigrosine negative staining and

Vlk (108), in 1938, confirmed the existence of mastigonemes with darkfield microscopy of living organisms. This light microscopy is discussed in detail in several reviews (16,

17, 93, 94). Early electron microscopy conclusively demon­ strated the reality of mastigonemes (17, 94).

The purpose of this dissertation is multifold. I will review the current and pertinent literature concerning the structure, taxonomic occurrence, assembly, and function of mastigonemes and describe the fine structure of the masti­ gonemes of two chrysophycean flagellates, Ochromonas minute and Monas sp. This will include the attachment of masti­ gonemes to and arrangement on the flagellum and their intra­ cellular development and transport to the cell surface. Some general features of the ultrastructure of the two organisms 2 will also be described. Observations on the structure, ar­ rangement and attachment of mastigonemes will be discussed in relation to flagellate motility in general. The insights into several basic problems in cell biology including self- assembly, functions of the nuclear envelope, modes of secre­ tion, Golgi apparatus function and dynamic membrane flow within the cell, will also be discussed. 3

LITERATURE REVIEW

Terminology and Mastigoneme Fine Structure

Bouck's definition of mastigonemes is used throughout this dissertation, that is to say, "any essentially fila­ mentous appendage that is clearly distinct from the flagellar membrane or its amorphous coverings" (12). Mastigonemes can be classified according to their struc­ ture into two major groups, the complex tubular mastigonemes and the fine fibrous mastigonemes. The features common to all tubular mastigonemes are: 1) a solid, tapering basal region 0.15-0.3 pm long which is often diffuse and rather ill-defined, 2) a tubular shaft 0.5-1.5 um long and 8-25 nm in diameter and 3) two to three fine terminal filaments

0.5-1.0 ym long. The major variables in tubular mastigoneme structure among taxa, that may be of some taxonomic value, are the overall length of the mastigoneme, the number of terminal filaments, the presence of a distinct basal region and the presence of filaments projecting laterally from the shaft. Nontubular or fibrous mastigonemes make up a structural­

ly diverse group. In general they are fine, flexible fila­

ments projecting from the flagellum in a disorganized array.

The arrangement of fibrous mastigonemes on the flagellum

varies from a precise helical array of tufts in some 4

Eugienophyceae (61, 82) to a "wooly" or tomentous coat in some Haptophyceae and Chlorophyceae (12). There is some ques­ tion whether the less structurally defined fibrous masti- gonemes found in some taxa are bonafide flagellar appendages or amorphous flagellar coatings precipitated into filaments during preparation for electron microscopy.

In some small marine flagellates with scaly flagella

(Prasinophyceae) Manton et al. (69, 74-78), and Parke and

Manton (89, 90) have reported flagellar hairs that do not appear to fit into either of the above two categories.

Coarse, often curved hairs with diagonal striations were re­ ported interspersed among the scales on the flagella of these organisms in shadowcast preparations. The attachment region was somewhat tapered although no distinct basal region was reported. Bouck (12) has suggested that if this description is confirmed with negative staining a third class of masti- gonemes may be indicated.

The subject of this dissertation is the complex tubular type of mastigoneme and the fibrous type will not be con­ sidered in any depth. The term mastigoneme when used below will refer to the tubular type unless otherwise stated. 5

Taxonomic Distribution

Mastigonemes have been reported in a wide variety of taxa of flagellated organisms. They are most prevalent in the algae which may possess both tubular and fibrous masti­ gonemes. Complex tubular mastigonemes have been reported in the algal classes Chrysophyceae (3, 4, 6, 1, 11, 13, 14, 17, 45, 58, 62, 94, 100), Xanthophyceae (23, 30, 62, 80), Eustigma- tophyceae (48), Cryptophyceae (12, 45), Bacillariophyceae

(12), Chloromonadophyceae (47) and Phaeophyceae (9, 10,

66, 72). Algal classes in which fibrous mastigonemes have been reported are the Dinophyceae (59, 60), Chrysophyceae

(11, 12, 13, 14, 53), Chlorophyceae (97) and Euglenophyceae

(61, 63, 82). The proper place for prasinophycean mastigonemes

in this scheme remains unclear. (See section above on Ter­

minology and Mastigoneme Structure ). In addition to these

algal classes, both tubular and fibrous mastigonemes have

been reported in the flagellated zoospores of some aquatic

phycomycetes (26, 37, 45, 76, 96). The only groups which

have both tubular and fibrous mastigonemes on the same

organism are the Chrysophyceae and aquatic phycomycetes.

Mastigonemes which appear to be of the fibrous type have

been reported in the bodonid flagellate Bo do saltans (15)

and in flagellated sponge cells (2). In general, complex tubular mastigonemes are most often found on the anterior 6 locomotory flagellum in heterokont flagellates and the smaller fibrous mastigonemes are found on the trailing flagel­ lum of these organism, on posterior flagella and on the anterior flagella of the Euglenophyceae and Chlorophyceae.

Bouck (12), in a recent review, has pointed out some general­ izations concerning the occurrence of mastigonemes. He noted that mastigonemes occur mainly in anteriorly or laterally flagellated vegetative or reproductive cells of "lower" organisms of several phyla, and that in biflagellate cells with one anterior flagellum, the mastigonemes on the trailing flagellum tend to be much reduced in size.

Differences in Mastigoneme Fine Structure Among Taxa

The Chrysophyceae have typical tubular mastigonemes with a tapering base (ca. 0.25 pm long), a shaft (ca. 1 ym long) and one to three terminal filaments (11-14, 45, 62). In addition, some of the Chrysophyceae have filaments projecting laterally from the mastigoneme shaft. Lateral filaments were not reported in Olisthodiscus (58, 62) or Giraudiopsis

(66) but Ochromonas danica apparently has two types of lateral O filaments (11). One type is short (300-400A) and radiates rigidly and perpendicularly from the shaft. The second type

is longer (3000Â) and appears more flexible. Lateral fila­

ments were not reported as such in Synura, Mallomonas or 7

Mallomonopsis, but Bradley (14) observed a fibrous mat around the mastigoneme shaft which may represent lateral filaments wrapped around the shaft. Xanthophycean mastigonemes are similar to those of the Chrysophyceae. The sizes of the base, shaft and terminal filaments are comparable; however; the base is less well defined and no lateral filaments are present (23 , 30 , 62 , 80). Two terminal filaments were re^- ported in all three xanthophycean species examined by Lee- dale et al. (62). The Phaeophyceae also have typical tri­ partite mastigonemes but no lateral filaments (9, 10, 66, 12).

Cryptophycean mastigonemes appear to be somewhat different. Heath and Greenwood (45) reported two sizes of mastigonemes on the two flagella of Cryptomonas; one type has a 1 ym shaft and the other a 2 ym shaft. Neither type has a taper­ ing basal region and both end distally in a single 0.7 ym

terminal filament. Bouck (12) found lateral filaments on

mastigonemes of Hemiselmis (Cryptophyceae) and these appeared shorter, thicker and more tortuous in shape than Chrysophyceae

lateral fibers. Heywood (47) reported that mastigonemes of

the chloromonadophycean Vacuolaria virescens have a taper­

ing base and tubular shaft but no terminal or lateral fila­

ments. This is the only report of mastigonemes without

terminal filaments. His observations were made exclusively on sectioned material however and final judgement on the

presence or absence of terminal filaments in this organism 8 must await a study of negatively stained whole mounted masti­ gonemes. The mastigonemes of the aquatic phycomycete zoo­ spores are very similar to the algal mastigonemes. They have a base, shaft and terminal filaments, but no lateral fila­ ments have been reported (37, 45, 76). In the past the arrangement of mastigonemes on the flagellum has been used as a taxonomic criterion (67, 68; see section on Arrangement of Mastigonemes on the Flagellum ) and it now appears that the structure of individual mastigonemes, especially the presence of lateral filaments, basal region and number of terminal filaments will prove of even greater phyletic value (12).

Arrangement of Mastigonemes on the Flagellum

The arrangement of mastigonemes on the flagellum appears to be precise and is probably critical to mastigoneme func­ tion (see section below on Mastigoneme Function ). On the anterior flagellum of the biflagellate Chrysophyceae masti­ gonemes are usually found in two bilateral rows (17, 58, 62).

Qlisthodiscus has two equal rows (5 8) but in Ochromonas danica one row is made up of a larger number of mastigonemes than the other (11). There are exceptions to the rule of two bilateral rows in the Chrysophyceae, for instance in

Pedinella a single row of mastigonemes attaches to a strength­ 9

ening band which runs up one side of the flagellum (104).

Deason (2 3) reported a helical disposition of mastigonemes in Pseudobumilleriopsis but no evidence was given. In the

Phaeophyceae, however, Bouck (10) has shown clear evidence

for helical rows of mastigonemes. In the flagellated zoo­

spores of the aquatic phycomycete Saprolegnia mastigonemes

are borne in two bilateral rows (76). Heath et al. (45)

reported that one flagellum in Cryptomonas (Cryptophyceae)

has a single row of short (1 ym) mastigonemes and the other

flagellum has two rows of longer (2 ym) mastigonemes. The

most common arrangement for mastigonemes on the flagellum

appears to be in two bilateral rows but variations and ex­ ceptions are not rare. The only class in which there is good

evidence for a helical distribution of tubular mastigonemes is the Phaeophyceae. An analysis of the flagellar action

of these organisms may help elucidate the functional signifi­

cance of such an arrangement since one would expect that dif­

ferences in arrangement of such structurally similar

appendages would be closely correlated to difference in

their mode of functioning.

Molecular Architecture of Mastigonemes

Data concerning the molecular arrangement within masti­

gonemes are accumulating but no general hypothesis has emerged.

Helical striations of 8 nm periodicity have been reported in 10 the tubular shafts of Olisthodiscus (Chrysophyceae) masti- gonemes (58, 62). Bradley (14) observed a periodic sub­ structure in the mastigonemes of Mallomonopsis (Chrysophy­ ceae) as did Leedale et al. (62) in Tribonema (Xanthophyceae) mastigonemes. Barton et al. (4) reported 42Â spaced globular subunits in staggered longitudinal rows in negatively stained

Ochromonas (Chrysophyceae) mastigonoxnes and he suggested that six such parallel rows would make up the wall of the tubular shaft of these mastigonemes. Bouck (10) suggested that the shaft of Ascophylum tubular mastigonemes were made up of ten rows of subunits with a 45Â center to center spacing. In Markham rotations of cross-sections of Sapro- legnia (aquatic phycomycete) mastigonemes, Heath and Green­ wood (45) found some reinforcement of five and six fold symmetry but many cross-sections showed no reinforcement.

Heywood (47) did Markham rotation studies on cross-sections of Vacuolaria (Chloromonadophyceae) mastigonemes and re­ ported eight-fold symmetry of the wall. He suggested that the wall of the mastigoneme shaft was made up of eight

4OA subunits. He also reported that the distal end of the tapering base of these mastigonemes is made up of a tubular wall with a central element in the lumen. This is the only report in the literature of substructure in the base.

Negatively stained bases of mastigonemes from many classes 11 of organisms do not appear hollow, and no geometric sub­ structure has been reported. Both Bouck (10) and Loiseaux and West (66) have described phaeophycean terminal filaments as single rows of globular subunits and have noted that two of these filaments frequently wrap around one another.

In summary, the present evidence suggests that masti- gonemes are made up of 1) a tubular shaft composed of 40-50Â globular subunits arranged in such a way that they can appear as five to eight longitudinal rows or as helical rows de­ pending on the penetration of negative stain, angle of sec­ tioning or other preparative variables, 2) an amorphous tapering base and 3) terminal filaments composed of a single row of globular subunits.

The only biochemical data available concerning mastigonemes are those of Bouck (11, 12) on Ochromonas mastigonemes. Isolated whole mastigonemes, when reduced, alkylated and electrophoresed on acrylimide gels show one major protein band. This indicates that there is a single major protein component in mastigonemes. Bouck (11) demon­ strated that this protein is different than the protein component of ciliary and flagellar microtubules. Periodic acid-Schiff (PAS) staining of the gels demonstrated addi­ tional carbohydrate bands. Since lateral filaments are added in the Golgi apparatus in Ochromonas (see section on Origin 12 and Assembly of Mastigonemes ) and since they are stable at 90°C for five minutes (12) Bouck suggested that they were the site of the carbohydrate moiety of mastigonemes. More data are necessary before a biochemical definition of mastigonemes is possible and differential solubilization and subsequent comparison of the electrophoretic patterns of these fractions with that of whole mastigonemes would be very enlightening in this regard.

Attachment of Mastigonemes to the Flagella

The nature of the attachment of the mastigoneme to the flagellum has been a matter of conflict in the literature.

Manton (67), Bradley (13, 14) and Deason (23) have reported that the mastigoneme base penetrates the flagellar membrane and attaches to the axoneme. These conclusions were based, for the most part, on whole-mount preparations of flagella in which the interpretation of whether the mastigoneme is passing under, over or through the flagellar membrane is at best difficult. Brown and Cox (17), Pitelka and Schooley

(94) and Bouck (10, 11) have reported mastigonemes attached to the flagellar membrane. Two convincing lines of evidence presented for this view are the adherence of mastigonemes to membrane fragments when the flagellum is disrupted, and micrographs of longitudinal sections through mastigoneme

bases where they insert on the flagellar membrane (Figures 13

8 and 9 in reference (10) and Figure 3 in reference (47)).

No similar micrographs have been published demonstrating mastigonemes passing through the flagellar membrane and inserting on the axoneme. In Ochromonas danica Bouck has found plate-like "attachment disc" modifications of the flagellar membrane at the site of mastigoneme attachment.

It is possible that the mastigonemes of some organisms at­ tach superficially while those of other organisms penetrate to the axoneme, but in light of their similarities in structure and origin (see section below on Origin and

Assembly on Mastigonemes ) this would seem unlikely.

Origin and Assembly of Mastigonemes

In the past few years several reports of the origin of mastigonemes in various taxa have been published and some generalities are now apparent. Mastigonemes develop in spaces enclosed by ribosome studded membranes, either the perinuclear space, plastid endoplasmic reticulum space or the cytoplasmic endoplasmic reticulum (6, 7, 10, 11, 23, 37, 40 , 58, 59 , 62). Bouck (12) and Gibbs (39, 40 ^ 41, 42) have emphasized that these three spaces are parts of a single continuous space in many lower flagellates and Bouck (11, 12) has called this space the perinuclear continuum. Mastigonemes developing within these spaces are usually attached by their 14 bases to the bounding membrane. There are, however, several differences in the mastigoneme developmental scheme among the taxa studied. These differences include: 1) the gross conformation of bundles of developing mastigonemes, 2) the structural completeness of the mastigonemes found in these spaces and 3) the involvement of the Golgi apparatus in mastigoneme development and transport.

In the Chrysophyceae (11, 45, 58, 62), the Xanthophyceae

(23, 62) and the aquatic phycomycetes (45) the developing or

"presumptive mastigonemes" (PMS, 10) are attached by their bases to the inside of the bounding membrane; this is usually

the outer nuclear membrane which bulges to form a bleb in

the perinuclear space. Often there is granular or amorphous

material within these spaces which may represent mastigonemal

precursor material (45, 58). What appears to be the

most mature vesicles or blebs in the perinuclear space con­

tain tightly packed presumptive mastigonemes usually present

in two antiparallel groups. This gives the vesicle a square-

ended appearance (11, 45, 58, 62). In the Phaeophyceae

(10) and Cryptophyceae (45) , vesicles containing presumptive

mastigonemes differ from the above in that the mastigonemes

are not aligned in a parallel fashion and associations of the bases with the bounding membrane cannot be detected. In these classes the presumptive mastigonemes are not so tightly

packed and appear to be in a rather random arrangement within 15 the vesicles (10, 45). In Vacuolaria (Chloromonadophyceae),

Heywood (47) reported huge bundles of presumptive mastigonemes in dilated cisternae of the endoplasmic reticulum. No mem­ brane associations were reported. These bundles occasionally ran the whole length of the organism (30-74 ym). They were so large and found so routinely that it was suggested that they may play a role in maintenance of cell shape.

Leadbeater (59) reported that fibrous mastigonemes in the dinoflagellate Woloszynskia micra develop in dilations of the perinuclear space also, so fibrous mastigoneme develop­ ment, in the Dinophyceae at least, appears to be somewhat similar to tubular mastigoneme development. However in

Euglenophyceae and Chlorophyceae, which have been extensively studied ultrastructurally (61, 63, 82, 97), there have been no similar reports, and the developmental sequence of fibrous mastigonemes in these classes remains obscure.

The second major difference in mastigoneme development among taxa involves the relative maturity or structural completeness of the presumptive mastigonemes found within the perinuclear continuum. In the Chrysophyceae (11, 45,

58, 62) and Xanthophyceae (23, 62) presumptive mastigonemes have tapering bases attached to the membrane, tubular shafts and terminal filaments. Aquatic phycomycete zoospores have bases and shafts but no terminal filaments (45). In the Phaeophyceae (10) and Cryptophyceae (45) only tubular shafts 16 are apparent in the perinuclear continuum. Tapering bases are known to be present in the mature mastigonemes of the

Phaeophyceae (10, 66), but when they are added or if they are present in the perinuclear continuum but were not detected is unknown. A similar situation exists in the

Chloromonadophyceae. Heywood (47) reported that only shafts were present in the dilations of the endoplasmic reticulum of Vacuolaria although mature mastigonemes on the flagellar surface and mastigonemes in a vesicle he observed being re­ leased at the cell surface were made up of both a shaft and a tapering base. In some chrysophyceans it has been reported that the cross-sectional diameter of the tubular shafts of presumptive mastigonemes is not as great as the diameter of mature mastigonemes on the flagellum (11, 58), suggesting that additional components may be added to a presumptive

mastigoneme before it is deposited on the flagellum. Bouck

(11) found that presumptive mastigonemes in the perinuclear

continuum of Ochromonas danica did not have lateral filaments

as do the mature mastigonemes; the addition of these ^la­

ments may explain the increase in cross-sectional diameter

that has been observed.

The third major variable in mastigoneme development in­

volves the participation of the Golgi apparatus in mastigoneme

development and transport. In Ochromonas danica, Bouck (11)

suggested that presumptive mastigonemes bleb off the peri- 17 nuclear space one or a few at a time in vesicles that are then added to the forming face of the Golgi apparatus. After these mastigonemes leave the Golgi apparatus, Bouck was able to de­ tect lateral filaments on their shafts. In light of what is known about the function of the Golgi apparatus in carbo­ hydrate metabolism (5, 83, 85), Bouck suggested that the lateral filaments were probably carbohydrate. Schnepf and

Deichgraber (100) reported tubules in the Golgi apparatus of

Synura (Chrysophyceae) which can now be recognized as

mastigonemes. There have been no other reports of Golgi apparatus participation in tubular mastigoneme development in any other taxa, although Leadbeater (59) and Leedale et al.

(62) have noted the proximity of the Golgi apparatus to the

site of presumptive mastigonemes in the Dinophyceae and

Chrysophyceae respectively. The dearth of reports of Golgi

apparatus involvement in mastigoneme development may be due

to the fact that this step is only necessary in organisms with mastigonemes that have lateral filaments, or Golgi

apparatus involvement may be a rapid and transient event

occurring only during actual flagellar growth and therefore

difficult to detect in normal cultures. For instance, Bouck

(11) observed Golgi apparatus associated mastigonemes only in

deflagellated cells that were actively regenerating flagella.

A thorough search for Golgi apparatus involvement in the Cryptophyceae, the only other algal class in which lateral 18 filaments have been detected (12), would be enlightening.

Manton et al. (78) have reported that the striated hairs found interspersed among scales on the flagella of Hetero- mastix (Prasinophyceae) develop in the Golgi apparatus along with these scales. These hairs have not been reported developing, at any stage, in the perinuclear continuum and membrane attachments in the Golgi apparatus have not been detected. The significance of this Golgi apparatus in­

volvement will remain unclear until more is known about the

fine structure of these flagellar hairs.

Release of Mastigonemes to the Flagellum

How mastigonemes find their way to the flagellum and be­

come deposited in the correct arrangement are problems that

are dependent on the mode of release of mastigonemes to the

cell surface. Bouck (10) suggested that the presumptive

mastigonemes of sperm developing in sperm concepticles of the

brown algae Fucus and Ascophylum, are extruded from the cell

and find their way by chance to "recognition sites" on the

flagellar membrane. The inefficiency of such a system with

free-swimming organisms is obvious. In Ochromonas danica,

however, Bouck (11) found mastigonemes attached to the

plasma membrane near the base of regenerating flagella. He

suggested that, after passage through the Golgi apparatus 19 and addition of lateral filaments, the Golgi apparatus de­ rived vesicles, containing one or a few mastigonemes, fuse with the plasma membrane near the base of the regenerating flagellum. The mastigonemes are then released by a secretory process with the membrane attachment of the base remaining intact. This leaves the new mastigoneme attached to the plasma membrane near the base of the growing flagellum where it can be "pulled up" the growing flagellum as new membrane

is required. Deason (23) suggested a similar mode of re­

lease for endoplasmic reticulum-derived vesicles containing

up to four mastigonemes which he observes near the cell

surface in Pseudobumilleriopsis (Xanthophyceae). An interest­

ing feature of such a mechanism is that while large amounts

of mastigonemes are assembled in a single space, the release

to the surface occurs in small numbers (1-4 mastigonemes/

vesicle). Apparently presumptive mastigonemes are removed

from the large assembly vesicle a few at a time as they are

needed, packaged and transported to the cell surface to be

released. Electron micrographs showing the actual transfer

of presumptive mastigonemes from the larger assembly vesicles

or dilations of the perinuclear space to the Golgi apparatus

have not been published. 20

Control of Mastigonemogenesis

Leedale et al. (62) , Deason (23) and Bouck (10) have noted that the occurrence of presumptive mastigonemes in the

Xanthophyceae and Phaeophyceae is dependent on the stage of the life cycle observed. Presumptive mastigonemes are found most consistently during the development of the flagellated stage of the life-cycle. The message to begin producing mastigonemes is evidently part of the genetic phase change that initiates a new stage in the life-cycle. In Ochromonas danica, Bouck (11) , found presumptive mastigonemes in the perinuclear continuum throughout the cell cycle, but in the

Golgi apparatus only in deflagellated cells which were re­ generating flagella. He suggested that "a rather precise feedback mechanism" existed among Golgi apparatus activity, mastigoneme assembly and flagellar length or growth. The assembly of mastigonemes in the perinuclear continuum appears

to be a rather constant process but the transport through the

Golgi apparatus and associated modifications occur only in

response to the need for mastigonemes for a new flagellum.

Mastigoneme Function

Fibrous and tomentous mastigonemes are generally believed

to enhance the efficiency of flagella by increasing their

surface area. Tubular mastigonemes, however, are usually 21 found on anterior flagella in which a planar sine wave is propagated anteriorly. The motion of organisms exhibiting this flagellar action is also anterior, so the thrust developed by the flagellum must somehow be reversed. Jahn et al. (52, 53) have presented a theory to explain how mastigonemes might function in this thrust reversal. They have suggested that the net thrust of mastigonemes moving backward against the media when the peak of the sine wave passes through the portion of the flagellum to which the mastigoneme is attached, is greater than the opposite thrust developed by the sine wave moving up the flagellum. They supported this hypothesis with the fact that polystyrene particles in the medium were propelled posteriorly as they came in contact with the peaks of the flagellar wave. Hol- will and Sleigh (50, 51), using the physical parameters of

Ochromonas flagella, modified the calculations of Taylor

(106) for the swimming of long narrow animals to show that such an hypothesis is hydrodynamically feasible. The amount of net forward thrust would be dependent on the rigidity and position on the flagellum of the mastigonemes. Hinged or semi-rigid mastigonemes would increase the thrust if they could move through 90° so that they projected perpendicularly from the flagellum when at the peak of the sine wave but laid down against the flagellum when in the trough of the wave. It is difficult to believe that the superficial mem­ 22 brane attachment could impart the necessary rigidity but the "attachment discs" reported by Bouck (11) or some sub- membrane modification might provide the necessary rigidity.

Holwill and Sleigh (51) pointed out that for the mastigonemes to efficiently reverse the thrust of the planar flagellar undulations, they must project from the flagellum in the plane of those undulations. It has been shown that the plane of flexure of a flagellum or cilium is perpendicular to the plane passing through the central pair of flagellar micro­ tubules (32, 38, 105). For mastigonemes to be effective they must therefore be attached to the flagellum in two bilateral rows perpendicular to the plane of the central pair of micro­ tubules. Bouck (11) presented three out of four micrographs of Ochromonas danica flagella showing mastigonemes attached in a plane parallel to the central pair. These results have been confirmed in this dissertation (see Figures 21a-e).

To date, the Jahn et al. hypothesis is still the best, but its inadequacies are becoming apparent and alternatives should be considered. In his recent review, Bouck (12) has suggested that although mastigonemes are not attached in the plane of the flagellar beat, they may "whip" from side to side and thereby project tangentially from the flagellum as the wave passes through it. Alternatively, there has been some evidence that the central pair does not define the plane of beat in all flagella. Fawcett (31) found a constant 20-30° 23 deviation of the central pair of flagellar microtubules from the plane perpendicular to the presumed plane of flagellar beat in guinea pig sperm. 24

MATERIALS AND METHODS

Culture Conditions

Monas sp. was isolated from a small pond on the Iowa

State University campus. It was grown in 0.1-0.2% (w/v) phosphate-buffered CerophylZ infusion (Cerophyl Labs) plus trace salts,^ inoculated heavily 24 hours previously with E. coli. A 10% inoculum using a two-day old Monas culture usual- ly gave late log phase cultures of 1-5 x 107 organisms/ml after two or three days at room temperature (21-24°C). Dif­ ferent lighting conditions had no effect on growth.

Ochromonas minute (Pringsheim, nom. prov.) was obtained from the Culture Collection of Algae at Indiana University,

Bloomington, Indiana (culture #L1300) and was grown axenical- ly on 0.1% (w/v) glucose, 0.1% (w/v) Tryptone, 0.1% (w/v) yeast extract and 0.2% (v/v) of a 1.5% (w/v) filtered liver extract infusion. Cultures were grown with daily swirling in 500 ml Delong culture flasks containing 100-150 ml of medium at 19-21°C under light of 600 ft-candles from 14 watt cool-white fluorescent tubes on a schedule of 16 hours light and 8 hours of dark. Samples for fixation and deflagellation were taken at mid- to late-log phase (5-10 x 10^ organisms/ml) two to three days after inoculation.

^See Appendix A. 25

Deflagellation and Kinetics of Reflagellation and Mastigoneme Transport

Both species could be effectively deflagellated (80-

90%) by forcing a suspension of organisms three to five times through a #22 hypodermic needle which had three 80-

90° bends. Reflagellation was initiated immediately upon removal from colchicine with several washes in growth medium.

Samples were taken at various times after deflagellation, colchicine treatment and removal from colchicine and prepared for electron microscopy as described below. At least 100 midnuclear sections which included nucleus, nucleolus and plastid were examined from each sample and the peri­ nuclear spaces (PNS) were classified as having either more than six, six or less, or zero presumptive mastigonemes

(PMs). At least 60 Golgi apparati from each sample were examined and scored as + or - for the presence of presumptive mastigonemes (PMs). The results of these counts are shown graphically in Figures 45 and 46.

Light Microscopy

Organisms were examined by phase-contrast and Normarski- interference-contrast optics on a Zeiss Photomicroscope and photographed in life using a Zeiss electronic flash unit

(Ukatron UN 60) and Kodak Plus X film. Chlorophyll auto-

fluorescence was tested using the Zeiss Photomicroscope with 26

HBO-200 Hg-vapor UV source, BG-12 exciter filter, bright-

field condenser and #44 and #47 barrier filters.

Electron Microscopy

Sectioned material

Samples were fixed for 1/2 to 1 hour in 2.5 or 4% (v/v)

glutaraldehyde (Fisher Biological Grade) in 0.1 M cacodylate-

HCl buffer at pH 7.2 with 0.01 M CaClg. The organisms were then washed in the same buffer without fixative but with

enough sucrose added to make the buffer isotonic to the fixa­

tive solution. The samples were then post-fixed in 1-2% (w/v) OsO^ in buffer for 2 hours at 4°C. Dehydration in an

ethanol or acetone series was followed in some cases with

the staining procedure of Locke et al. (64; 60°C for 12

hours in 1% (w/v) uranyl acetate in absolute ethanol).

All material was embedded in either Maraglas (102) or Spurr's

low-viscosity ERL (103). Sections were cut with a Dupont

diamond knife on an LKB Ultratome 1, picked up on parlodion-

carbon coated copper grids and stained with 2% (w/v) uranyl

acetate in water or 50% (v/v) ethanol followed by 0.2-0.3%

(w/v) alkaline lead citrate (107). Those previously treated

with the procedure of Locke et al. (64) required no addition­

al uranyl acetate staining. 27

Whole mount material

A drop of the suspension of organisms in either growth medium or distilled water if osmotic disruption was desired, was placed on a coated grid and the particles allowed to settle. After a few minutes the excess fluid was removed with filter paper. Grids were examined directly or negative­ ly stained with either 1% (w/v) phosphotungstic acid (PTA) at pH 6.0 or 1% (w/v) uranyl acetate. Staining was ac­ complished by washing with several drops of the staining solu­

tion and removing the final drop with filter paper until a thin film of stain remained on the grid.

Microscopy

All grids were examined on either an RCA EMU 3-F elec­

tron microscope operating at 50 or 100 KV or an Hitachi

HU-ll-E-1 electron microscope operating at 50 KV. Some of

the statistical data were obtained by examining on the fluores­

cent screen every favorable organism in each grid square,

first at low magnifications (3,500X), then at high magnifi­

cations (10-50,OOOX) to evaluate presence or absence of

presumptive mastigonemes. 28

RESULTS

General Morphology

Figures 6, 7, 16, and 17 are phase-contrast flash micrographs of Monas and 0. minute. The organisms are structurally quite similar, differing primarily in size,

(Monas is 4-7 ym long and O. minute is 10-12 ym long) and structure of the plastid. The Monas plastid is smaller and contains only limiting membranes while the 0. minute plastid contains a few intra-plastid thylakoid membranes

(Figures 10, 18, 19, 29, 31, 43). Using the criterion of autofluorescence, 0. minute plastids were found to contain chlorophyll while Monas was chlorotic. This observation was corroborated by the appearance of cultures of 0. minute which were brown-green while those of Monas were colorless.

Both organisms have an anterior contractile vacuole and a nucleus with a single central nucleolus. Although not demon­ strated in this particular selection of light micrographs,

there is often a large leucosin vacuole filling the posterior

of the organism, especially in O. minute. The plastid is

always found adjacent to the nucleus, extending to the surface

of the organism near the base of the short flagellum where

the eye-spot is found. The single plastid in these organisms

is enclosed within an invagination of the outer nuclear mem­

brane and therefore becomes surrounded by two thicknesses of 29 the outer nuclear membrane. The plastid then does not lie in the perinuclear space as it appears in some micrographs, but is separated from that space by the inner of the two layers of outer nuclear membrane. This situation is not as clear in micrographs of O. minute and Monas as it is in

O. danica (11, 40-42) since the outer chloroplast membrane and the inner of the two layers of outer nuclear membrane tend to be closely opposed.

The close association of the single Golgi apparatus with the nuclear envelope is also evident (Figures 18, 19). The mitochondria, which are more numerous anteriorly, have tubular cristae and a dense matrix. 0. minute may contain a con­ siderable amount of rough endoplasmic reticulum; Monas has none except the outer nuclear membrane. Lipid droplets stain very intensely in organisms treated with hot ethanolic uranyl acetate prior to embedding (Figures 18, 19).

Mastigoneme Structure, Arrangement and Attachment

Both organisms have typical chrysophycean tubular mastigonemes made up of a proximally tapering base 0.25 ym long by 0.03 ym wide at the distal widest point; a hollow shaft 0.85 ym long by 2 3 nm wide attached to the distal end of the base; terminal filaments and two types of lateral filaments (Figures 3, 4, 5, 24, 25). The short lateral 30 filaments (30 nm long) project from the shaft at about a 30° angle giving the shaft a "fuzzy" appearance. The long lateral filaments may be up to 0.5 ym long and are fairly regularly spaced along the shaft at 50-80 nm intervals. The mastigoneme bases lack lateral filaments.

The precise molecular architecture of the mastigoneme shaft remains undetermined. In some negatively stained prep­ arations helical and longitudinal rows of subunits can be seen

(Figure 5). In this figure the longitudinal center to center spacing of these subunits is 5-6 nm; the transverse center to center spacing is 7-8 nm. The longitudinal rows are more apparent in negatively stained presumptive mastigonemes from dilations of the perinuclear space (Figure 32), and the center to center spacing here is also 7-8 nm. The lateral filaments appear as single rows of subunits. The tapering bases appear somewhat fibrous and no subunit structure can be recognized.

Figures 1 (Monas) and 20 (0. minute) are unstained whole mounts of intact organisms showing two unequal rows of mastigonemes on opposite sides of the long flagellum. One row is made up of "tufts" of 2-4 mastigonemes attached to thickened modifications ("bumps") of the flagellar membrane at 0.2-0.3 ym intervals. From several micrographs of this type it has been determined that there are approximately 20 31 mastigonemes per ym of 0. minute flagellum. Figures 21 A-E are cross-sectional micrographs of long flagella at the level of mastigoneme attachment showing the relationship of mastigonemal rows to the axoneme. They indicate that the mastigonemes join the flagellum in a plane which passes through both of the central pair of axonemal microtubules.

Fibrous mastigonemes are present on both the long and short flagella (Figures 4, 5, 24), but because of the absence of tubular mastigonemes, they are better illustrated on the short flagellum (Figure 1).

In disrupted flagella (Figures 3, 4, 24, 25), the mastigonemes remain adherent to flagellar membrane fragments indicating that this is their primary site of attachment.

The areas of the membrane to which they are attached are often somewhat thickened and plate-like (Figures 2, 21, 22, 23).

While there is a modification of the membrane at the attach­ ment site and some sub-membrane specialization, (Figures 2,

22, 23) we have seen no indication that mastigonemes connect directly to the axonemal microtubules.

Mastigoneme Morphogenesis and Transport to the Flagellum

Presumptive mastigonemes (PMs) are found in dilations of the perinuclear space (Figures 8, 9, 10, 26, 27, 2 8).

Dilations containing a few unoriented PMs and much granular 32 amorphous material, which may represent a mastigoneme pre­ cursor, are assumed to be immature (Figures 8-10). PMs are attached to the inside of the outer nuclear membrane by the narrow proximal end of their bases (Figures 11, 30).

This attachment can be found in some of the earliest stages

(Figure 8) suggesting that PM assembly may begin with the base. As PMs mature within the perinuclear dilations, they become organized in a more parallel fashion, filling the dilation and giving it a square-ended appearance (Figures

11, 12, 29, 30). In such dilations there may be a single group of parallel PMs (Figure 11), or more often, two anti- parallel groups of PMs with their bases attached to the membrane at opposite ends of the dilation (Figures 29, 12).

Figure 32 shows a negatively stained whole mount of a bundle of PMs contained within the perinuclear dilation of an osmotic- ally disrupted organism. When compared to the structurally mature mastigoneme in the lower left quadrant of this micro­ graph, several interesting differences are noted. First, no lateral filaments are present on the PMs, making the shaft look thinner (18 nm vs. 2 3 nm) and also revealing substructure in the shaft that is obscured by the lateral fila­ ments in structurally mature mastigonemes. The most frequent substructural pattern appears as three longitudinal striations.

Another difference is that the mature mastigoneme base is distinct and intact while in the PMs, the basal region is 33 seen only as a mass of fibrous material. In only one case

(Figure 32, arrow) has the base retained any integrity.

From thin-sectioned material it can be seen that the bases of PMs at this stage are in fact well formed (Figures 11,

30). Apparently the mature base is unaffected by the osmotic shock treatment, PTA treatment, and drying, while the im­ mature base is disrupted by one or a combination of these treatments. This indicates that the basal structure is some­ how stabilized at a later stage in the developmental sequence.

Terminal filaments are present on both mature mastigonemes and

PMs. Figures 14 and 15 (Monas) and 35-38 (O. minute) show the occurrence of PMs in the Golgi apparatus. Close associations between mature perinuclear bundles of PMs and the Golgi apparatus have been observed in cells rapidly regenerating flagella (Figures 33, 34) but the mechanism of transport of

PMs a few at a time from these bundles to the Golgi apparatus is unclear. Golgi apparatus PMs are seen less frequently in normal log phase cells than in cells regenerating flagella where they are found as singlets, doublets and occasionally as triplets and quartets in the dilations at the peripheral extremities of Golgi cisternae.

Figures 39-42 show mastigonemes being released from

Golgi-derived vesicles to the cell surface by what appears to be a secretory process. The membrane association of the 34 mastigoneme remains intact throughout the release process so they are positioned on the cell surface near the base of the growing flagellum (Figures 40, 43, 44). It appears that as the axoneme lengthens, the membrane around its base to which the newly emerged mastigonemes are attached, becomes part of the flagellar membrane. Thus the mastigonemes arrive on the flagellar surface.

The asymmetry of distribution of mastigonemes appears to be established at a relatively early stage in the develop­ mental process. The segregation of single mastigonemes and small groups destined to be "tufts" can be seen in the Golgi apparatus in Figure 35.

Analysis of the Intracellular Location of Presumptive Mastigonemes During Flagellar Regeneration and Colchicine Inhibition

Mastigoneme formation and transport in Monas and Ochro- monas, while showing many similarities to classical Golgi apparatus exocrine function, appear to be a more complex series of events involving several components and more pre­ cise origin and destination sites of the secreted product.

This system lends itself to a quantitative analysis of the numbers of presumptive mastigonemes at the various stages of development at various times during flagellar growth as a means of assessing the transport of presumptive mastigonemes from the nucleus through the Golgi apparatus to the flagellar surface. Figures 45 and 46 are graphs showing the results of 35a these quantitative studies on the occurrence of PMs in the perinuclear space and Golgi apparatus of 0. minute at various times after deflagellation, colchicine inhibition and re­ lease from colchicine inhibition. Reflagellation kinetics are plotted in curve A in Figures 45 and 46, and are similar to those reported by Rosenbaum and Child for 0. danica (98) in having a lag period followed by decelerating growth. We noted a much faster maximum growth rate, however, (0.15 ym/ min vs. 0.0 8 um/min) which may be due to species differences, method of deflagellation, or sampling methods (monitoring of living, reflagellating cells on a microscope slide vs. observing samples fixed at various times after deflagella­ tion). Curve B in Figure 45, shows that the occurrence of large bundles of PMs in the perinuclear space (> 6 PMs/PNS) decreases in a sample of cells rapidly regenerating flagella, while small bundles (curve C, < 6 PMs/PNS) and the percentage of Golgi apparatus with PMs (curve D, % Golgi c PMs) increases.

After one hour of reflagellation, the number of perinuclear spaces with large numbers of PMs begins to increase again. Then as flagellar growth slows, the number of small bundles of PMs and of Golgi apparati containing PMs decreases.

In Figure 46, arrow #1 indicates the time of deflagella­ tion and colchicine administration, and arrow #2 indicates the

time of release from colchicine by washing several times with

growth media. This graph shows that deflagellation, even in 35b the presence of colchicine, initiates changes in the observed location of PMs, but that after a short period of time (less than 15 minutes, sample 15Ch) these changes cease and a static condition is maintained until colchicine is removed and flagellar growth occurs. The first reaction to this flagel­

lar growth is the rapid disappearance of PMs from the Golgi

apparatus; subsequently the PMs follow a pattern very simi­

lar to the simple deflagellation case shown in Figure 45. Figures 1-5. Electron micrographs showing the arrangement, insertion and fine structure of Monas sp. mastigonemes

1. Unstained whole-mount of Monas sp. showing the long flagellum (LF) with its asymmetri­ cal, bilateral array of tubular mastigo­ nemes (M) and the short flagellum (SF) with fibrous mastigonemes (FM) (line scale = 1.5 vim) 2. Section through the long flagellum showing the mastigoneme attachment and associated membrane modifications (arrows) (line scale = 0.5 ym)

3. Negatively stained (1% PTA) disrupted flagellum showing the tapering base (b), tubular shaft (s) and lateral filaments (If) of the mastigonemes. The mastigoneme base (b) inserts on a membrane fragment (m) (line scale = 0.5 ym)

4. Same as Figure 3 showing fibrous mastigq- nemes (FM) on the disrupted long flagellum (line scale = 0.5 ym)

5. Negatively stained intact flagellum showing tubular mastigonemes with terminal fila­ ments (tf) and both long (arrow #1) and short (arrow #2) lateral filaments. Longi­ tudinal rows of subunits can be seen with­ in the shaft at several points (white arrows) (line scale = 0.5 ym) 37 Figures 6-7. Phase-contrast, flash light micrographs of living Monas sp.

6. Unflattened organism showing the size of Monas sp. and the position of the long and short flagellum during swimming (line scale = 10 ym)

7. Flattened organism showing long and short flagellum, nucleus with central nucleolus, contractile vacuole and small plastid (P) (line scale = 10 urn)

Figures 8-10. Electron micrographs of thin sections through the nucleus (N) of Monas sp. showing early stages of presumptive mastigoneme (PM) de­ velopment

8. Two dilations of the perinuclear space (PNS) , one contains granular amorphous material and a few developing mastigonemes attached to the outer nuclear envelope (arrow) and the other contains pre­ sumptive mastigonemes (PM) (line scale = .0.5 ym)

9. An immature perinuclear dilation with granular amorphous material and few pre­ sumptive mastigonemes (arrows) (line scale = 0.5 ym)

10. Relationship between nucleus (N), nucleo­ lus (n) , perinuclear space (PNS) , plastid (P), eye-spot (ES) and swelling of the short flagellum (FS). Note the absence of internal membranes in the plastid and the attachment of developing mastigon­ emes to the outer nuclear membrane (arrow) (line scale =0.5 ym)

Figures 11-15. Electron micrographs of thin sections of Monas sp. showing late stages of presump­ tive mastigoneme (PM) assembly and transport

11. Perinuclear dilation containing a single parallel group of presumptive mastigone­ me s (PM) attached by their bases to the outer nuclear membrane. The nucleus (N) is shown (line scale = 0.5 ym)

12. Perinuclear dilations on two sides of the nucleus (N) containing bundles of presumptive mastigonemes (PM), one in cross-section with more than two dozen PMS and the other in longitudinal section containing two antiparallel groups of presumptive mastigonemes at­ tached at opposite ends of the peri­ nuclear dilation (line scale = 0.5 um)

13. A, B, and C. Three serial sections through a mature bundle of presumptive mastigonemes which is free in the cyto­ plasm showing bases (b) and shafts (s) (line scale = 0.5 um)

14. Golgi apparatus containing an obliquely sectioned presumptive mastigoneme (arrow) (line scale = 0.5 ym)

15. Golgi apparatus containing two cross- sectioned presumptive mastigonemes (arrow) (line scale =0.5 ym) 41 Figures 16-17. Phase contrast, flash, light micrographs of flattened, living Ochromonas minute showing the nucleus, nucleolus (n), plas- tid (P), eye-spot (ES), contractile vacuole (CV), short and long flagella (line scale = 10 um)

Figures 18-19. Survey electron micrographs of 0. minute 18. 0. minute, 15 minutes after deflagella- tion, showing short flagellum, re­ generating long flagellum, nucleus (N), nucleolus (n), plastid (P) with eye- spot (ES) and the Golgi apparatus, which contains a presumptive mastigoneme (arrow) (line scale = 10. ym)

19. The relationship between the nucleus, plastid (P) and Golgi apparatus. Note the bundle of presumptive mastigonemes (PM) in the perinuclear space and the osmiophilic lipid droplets (LD) (line scale = 10. um) 43 Figures 20-25. Electron micrographs showing the arrange­ ment/ attachment and fine structure of mastigonemes of 0. minute

20. Unstained, whole-mount of the long flagellum (LF) showing the asymmetric, bilateral array of mastigonemes. Note tufts of mastigonemes on the right side of the flagellum (line scale = 1.0 ym)

21. A-E. Cross-sections through five flagella showing the attachment of the mastigonemes relative to the central pair of axonemal microtubules

22. Longitudinal section through the long flagellum showing the attachment of mastigonemes and associated membrane modifications (arrows) (line scale = 0.5 ym)

23. Same as Figure 22. Arrows show mem­ brane thickenings at the sites of mastigoneme insertion (line scale = 0.5 ym)

24. Negatively stained (1% PTA) mastigone­ mes from a disrupted flagellum show­ ing the structural components of the tubular mastigonemes, their attachment to membrane fragments (m) and the presence of fibrous mastigonemes (FM) (line scale = 0.5 ym)

25. Same as Figure 24 showing the tapering base (b) attached to membrane frag­ ments (m), the tubular shaft (s) and lateral filaments (If) (line scale = 0.5 ym) 45 Figure 26-31. Electron micrographs of thin sections of 0. minute showing develop­ mental stages in presumptive mastigoneme assembly. Nucleus (N)

26. An early stage in presumptive mastigoneme development in the perinuclear space. Note the granular amorphous material and few formed presumptive mastigonemes in the perinuclear dila­ tion (line scale = 0.5 ym)

27. Same as Figure 26 (line scale = 0.5 ym)

28. A more mature bundle of presumptive mastigonemes in the peri­ nuclear space (line scale = 0.5 ym)

29. A dilation of the periplastid space (chloroplast endoplasmic reticulum) containing presumptive mastigonemes (line scale = 0.5 ym)

30. A bundle of presumptive mastigonemes in the perinuclear space that have distinct tapering bases (b) (line scale = 0.5 ym)

31. A rough membrane bound vesicle containing presumptive mastigo­ nemes found free in the cytoplasm away from the nucleus (N) (line scale = 0.5 ym) 4 6b Figure 32a,b. Negatively stained whole mount of (32a) a bundle of PMs. from an osmotically disrupted 0. minute and (32b) a mature mastigoneme from the same preparation. The mature mastigoneme has a base (b), shaft (s), and lateral filaments (If). The terminal filaments can not be seen on this specimen. The PMs have tubular shaft (s) and terminal filaments (tf), but lack lateral filaments and structurally intact bases. The only remnant of a base is shown at the arrow. The PMs are narrower than the mature mastigoneme and longitudinal striations can be seen more clearly. Nuclear membrane (m) (line scale = 0.5 ym) 47b Figures 33-34. Electron micrographs of sections through the Golgi apparatus of O. minute showing the transfer of presumptive mastigonemes from the perinuclear space to the Golgi apparatus. Nucleus (N)

33. Organism 15 minutes after deflagella- tion showing the close association of a bundle of mastigonemes with the Golgi apparatus (line scale =0.5 ym)

34. Same as Figure 33. Organism ten minutes after release from colchicine inhibition of flagellar regeneration (line scale = 0.5 um) 49 Figures 35-38. Electron micrographs of sections showing presumptive mastigonemes in the Golgi apparatus of 0. minute. Nucleus (N)

35. Presumptive mastigonemes in the lateral dilation of Golgi cisternae (arrows) in an organism with a growing flagel- lum (line scale =0,5 ym)

36. Organism 15 minutes after deflagella- tion with presumptive mastigonemes in the lateral dilations of the Golgi apparatus. Note the presumptive mastigonemes seem to be segregated into groups of two or more in the upper lateral dilations and singlets in the lower dilations (line scale = 0.5 ym)

37. Longitudinal section through pre­ sumptive mastigonemes (two per vesicle) in the Golgi apparatus near the base of a growing flagellum 60 minutes after deflagellation (line scale = 0.5 ym)

38. Longitudinal section through a pre­ sumptive mastigoneme associated with the Golgi apparatus (arrow) of a colchicine inhibited deflagellated cell showing the attachment of the tapering base to the Golgi cisternae membrane (line scale = 0.5 ym)

Figures 39-44. Electron micrographs of thin sections of 0. minute showing the release of presumptive mastigonemes to the cell surface 39. The periphery of a cell 120 minutes after deflagellation showing a mastigoneme with lateral filaments in a vesicle at the edge of the Golgi apparatus and very near the cell surface (line scale = 0.5 ym)

40. Organism 30 minutes after deflagella­ tion, showing three presumptive mastigonemes (arrows), one in the Golgi apparatus, one in a vesicle be­ tween the Golgi apparatus and the cell surface and one on the cell surface attached to the plasma mem­ brane (line scale = 0.5 ym)

41. The apparent release of mastigonemes to the cell surface by exocytosis (line scale = 0.5 ym)

42. Same as Figure 41. Organism 60 minutes after deflagellation (line scale = 0.5 ym)

43. Section through the kinetic apparatus showing the association of the eye- spot (ES) with the short flagellum and mastigonemes on the cell surface at the level of the basal body (bb) of the long flagellum (arrows) (line scale = 0.5 ym)

44. Lack of flagellar growth after 60 minutes in colchicine post deflagella- tion. Mastigonemes however, have been deposited on the cell surface near the flagellar stub (arrow) and on the flagellar stub itself (line scale = 0.5 ym) 53 Figure 45. Results of counts made on thin sections of 0. minute fixed at various intervals after deflagellation Curve A. Reflagellation reference curve determined from light micro­ scopy of living cells. Flagellar length in pm is shown on the right-hand Y-axis. The X-axis represents minutes after deflagellation

Curve B. Solid circles, > 6 PMs/PNS. The percentage of mid-nuclear sections, including nucleolus and plastid, which had more than six presumptive mastigonemes in the perinuclear space (left Y-axis) plotted against time after deflagellation (X-axis). The first points in each curve, plotted above C on the X-axis, were determined from a control sample of un- deflagellated, log-phase cells Curve C. Open circles, < 6 PMs/PNS. The percentage of mid-nuclear sections (same criterion as for curve B), that contained from 1-6 presumptive mastigonemes in the perinuclear space, plotted against time after deflagellation

Curve D. Squares, % Golgi with PMs. The percentage of the Golgi apparati which were observed to contain at least one presumptive mastigoneme, plotted against time after deflagellation •->6PM's/n>ii 0-<6PMs/PNS lOpm •- %Golgi c PM's

Sjim

en LT. ÔMm

4pm

- 2 pm

15 . 30 minutes Figure 46. Results of counts made on cells deflagellated in colchicine, left on colchicine for one hour and then washed several times and returned to growth media

Curves A,B,C and D have the same meanings as in Figure 45

Arrow #1. The time of deflagellation in colchicine

Arrow #2. The time of removal from colchicine

Points on the X-axis, samples;

Sample C. A control sample of log-phase cells

Samples 15Ch and 60Ch. Indicate minutes after deflagellation in colchicine

Samples 10, 20, 45 and lOOpCh. Indicate minutes after removal from colchicine 60 #- >6PM/PNS O- <6PM/PNS 2 • - %Golgi F PM's

Sum"

40 \ Ln %

20

mJJt C 15Ch 60Ch lOpCh 20pCh 45pCh lOOp Fig.46 minutes Ch 58

Figure 47. Schematic drawing of postulated origin, develop­ ment, and attachment of mastigonemes in 0. minute. PM bundles, labeled 1 and 5, are groups of pre­ sumptive mastigonemes (PMs) which are formed in the perinuclear space and transported through peripheral Golgi vesicles to the cell membrane and thence to the flagellar membrane.' PM bundles appear to be differentiated to the extent that PMs move to the Golgi one at a time from bundle 1 and in groups of two or three (tufts) from bundle 5, with subsequent movement to the cell surface probably controlled by the Golgi appara­ tus. This transport is labeled 2-4 for single PMs and 6-8 for those destined to be tufts (P = plastid) 59

DISCUSSION

Phyletic Relationship of Monas to O. minute

O. minute and Monas sp. are very similar ultrastructur- ally, differing primarily in the size and complexity of the plastid. This obvious similarity suggests that Monas is the

colorless analog of Ochromonas just as Polytomella is to Chlamydomonas. There appears to be a stepwise phyletic as­

cent through the ochromonad flagellates as demonstrated by

size; photosynthetic capabilities, plastid size and amount

of internal membrane; complexity of culture media required

and phagocytic activity. The general structure and organelle

arrangement of these organisms appear to be quite similar.

Ochromonas danica (10, 11, 40) is largest (15 ym) and it has

a large plastid. It will grow readily as a photoautotroph

(1) and its phagocytic activity is generally low. O.

malhamensis (1, 95) is smaller and less photosynthetically

active. 0. minute is still smaller and has a small plastid

with internal membranes. I was unable to grow it as a

photoautotroph and it required a rich media for abundant

growth (see Materials and Methods). Monas sp. is extremely small (4-7 ym) with a small chlorotic plastid. It could not

be readily cultured axenically and is a very active phago­

cyte. 60

A close relationship is further supported by the identi­ cal mastigoneme structure and similar development in the two organisms studied here and O. danica (11). However, there were differences; for example, in Monas large mature peri­ nuclear PM bundles were prevalent, while in O. minute, they were rare. On the other hand, small perinuclear bundles and

Golgi apparati with PMs occur more frequently in O. minute than in Monas. Perhaps differences in cultural conditions and growth rate could account for this effect. Since Monas was growing slowly, there was time for excess PMs to accumu­

late and form large perinuclear bundles, while the more

rapidly growing 0. minute utilized mastigonemes as fast as

they were produced. This could also account for the seeming­

ly energy expensive process of concentrating the PMs into

highly organized vesicles only to peel them off one or a

few at a time for use. The large, mature bundles are probably

for efficient storage of excess PMs for future use.

Arrangement of Mastigonemes Relative to their Function

Jahn et al. (52, 53) in one of the best attempts to ex­

plain mastigoneme function (see Literature Review, section

on Mastigoneme Function), suggested that they act in reversing

the thrust of the flagellar wave. This requires that

mastigonemes, if in a planar arrangement, be attached in the 61 same plane as the flagellar flexure. It is commonly accepted that in planar beating cilia and flagella, the plane of flexure is at right angles to a plane intersecting both of the central pair of microtubules (30). Therefore, to function according to

Jahn's theory, mastigonemes must be attached at right angles to this central pair plane also. Bouck (11) showed three out of four micrographs of flagellar cross-sections of O. danica which indicated that mastigonemes are attached to the flagellum close to the central pair plane; i.e., 90° opposed to the attachment plane required by the theory. Our results with Monas and 0. minute (Figures 21 A-E) confirm the sugges­ tion of Bouck. It thus appears that either the plane of flagellar flexure in the ochromonad flagellum is abnormal or that the Jahn et al. theory needs modification. The rela­ tion of the asymmetric distribution of mastigonemes on the flagellum to flagellar function is unknown.

Attachment of the Mastigoneme to the Flagellum

The adherence of mastigonemes to membrane fragments in disrupted flagella was observed many times during this study

(Figures 3, 24, 25); however, in longitudinal sections through the flagellum at the level of mastigoneme attachment, regional modifications of the membrane were clearly visible and some subsurface structural component was also apparent (Figures 2, 62

22, 23). These probably correspond to the "disc-like modi­ fications" of the membrane reported by Bouck (11) at the site of mastigoneme attachment in 0. danica. One can imagine how these membrane thickenings could be mistaken for the

base of the mastigoneme penetrating to the axoneme. We are of the opinion that the mastigoneme, as defined, is essential­

ly external and any submembrane components are accessory to

and not strictly a part of the mastigoneme. It is likely,

however, that this structural modification of the membrane

is introduced at the attachment site at some early stage in

mastigoneme development and subsequently carried to the

flagellum on the membrane as is the mastigoneme. Figures 11,

12, 28, 29, 30, and 38 demonstrate that the region of the

outer nuclear membrane where the bases of large bundles of

presumptive mastigonemes attach, appears thick and somewhat

rigid. This rigidity may help to impart the square-ended

appearance to these chambers. The thickenings of these mem­

branes may correspond to and eventually become the membrane

modifications seen at the site of mastigoneme attachment to

the flagellum.

Intracellular Development, Transport and Release of Mastigonemes

The sequence of events in the assembly, transport and release of mastigonemes that we propose from this study is

similar to that suggested by Bouck (11) for O. danica. It 63 begins with the development of PMs in the perinuclear space

presumably from amorphous precursors by primarily self-

assembly processes. There may be a nucleation site on the

membrane which initiates the assembly of the base which would

in turn initiate the shaft assembly. In this way information

could be carried on the membrane to orient the PMs correctly

in the perinuclear dilation. If the organism has a low de­ mand for mastigonemes, the dilations of the perinuclear space may become packed with many parallel PMs. These dila­

tions may pinch off and become free vesicles in the cyto­

plasm. If, on the other hand, the demand for mastigonemes

is great, as in cells regenerating flagella, the perinuclear bundles of PMs do not become very large since they are being

drained rapidly to supply the new flagellum. The method of

transfer of PMs from the perinuclear space to the Golgi

apparatus is unknown. They appear to be transferred one or a

few at a time to the lateral dilations of the Golgi cisternae.

Bouck (11) found PMs in the Golgi apparatus only of cells re­

generating flagella; in our study, although such cells showed

an increased incidence of Golgi PMs, we also found that a

significant percentage (13%) of the sections through the Golgi apparatus of log phase control samples demonstrated

PMs. 64

Golgi apparatus function

The Golgi apparatus has been shown in many systems to be involved in the addition of carbohydrate to protein (5, 83,

85, 112, 114). Bouck (11, 12) has determined that there is a carbohydrate moiety in Ochromonas mastigonemes and has sug­ gested that, since lateral filaments are added in the Golgi apparatus, they may be the site of mastigoneme carbohydrate.

In this study, I have shown that in addition to lateral fila­ ments, the base is modified in the Golgi apparatus, suggesting indirectly that it may also acquire some carbohydrate. Carbo­ hydrate polymers are generally resistant to environmental stresses and are often utilized biologically as protective cell coverings; e.g., cellulose cell walls. One of the func­ tions of the carbohydrate moiety of mastigonemes may likewise be to protect the proteinacious mastigoneme from its hostile extracellular environment. This protective function is sug­ gested by the relatively greater stability of the base of mature mastigonemes to negative staining procedures than the bases of PMs found in the perinuclear space. The position of the lateral filaments, external to and coating the mastigoneme shaft, makes a protective function for them feasible also. These suggestions are speculative and the confirmation of the biochemical nature of mastigonemes awaits careful fractionation and biochemical analysis. With respect to the study of Golgi apparatus function, mastigoneme develop- 65 ment offers a readily available model system with morphological markers. The packaging and carbohydrate metabolism functions of the Golgi apparatus which are defined biochemically in a variety of systems (the secretion of mucus, enzymes, extraneous cell coats) can be studied at the morphological level.

A second general function of the Golgi apparatus is the control of the nature of the cell surface (112; e.g., the production of algal cell walls, 18-20, 71; duodenal cell coats, 8; Amoeba cell coats, 114; fungal cysts, 46; and algal scales and coccoliths, 69-71, 74 , 78, 88, 113). This Golgi apparatus function is apparent in the transport and release of mastigonemes to the cell surface. Not only does the Golgi apparatus play a role in determining the chemical nature of the flagellar surface (e.g., the biochemical make up of the mastigoneme in this case), but also seems to be involved in the production of a precise spatial arrangement of mastigonemes on the surface of the flagellum (see section on Control of Mastigoneme Arrangement).

Recently there have been several reports suggesting a net flow of membrane through various membraneous organelles of the cell (21, 22, 29, 30, 43, 44, 86, 110). This hypoth­ esis generally states that membrane, arising from the nuclear envelope or endoplasmic reticulum, moves as small vesicles to coalesce at the "forming face" of the Golgi apparatus (84). As membrane is added at this face, it is being lost from the 66

"mature face" as secretory vesicles or lysosomes which, in turn, fuse with the plasma membrane or phagocytic vacuoles.

Evidence in support of this hypothesis includes the inter- mediacy of the Golgi apparatus membranes between endoplasmic reticulum and plasma membranes, in biochemical composition

(55), enzymatic activity (21, 22), and histochemical reac­ tivity (115). There is also a morphological gradient across the Golgi apparatus from nuclear envelope-like to plasma membrane-like (44). In addition it has been shown in some species that if the nucleus is removed from the cell, the

Golgi apparatus will soon disappear (35, 36, 110). This evidence and electron micrographs of vesicles which appear to be blebing off from the nuclear envelope or adding to the forming face of the Golgi apparatus (26, 30, 96, 110) suggest the dynamic nature of membraneous organelles, but actual net flow of membrane has not been demonstrated. The fact that mastigonemes are attached to the membrane throughout their development and can be found in a temporal sequence during re- flagellation, first in the perinuclear space attached to the nuclear envelope, then in a more structurally mature state

(i.e., with lateral filaments) in the Golgi cisternae attached to the Golgi membranes and finally on the cell surface at­ tached to the plasma membrane, is much more compelling evi­ dence that a net flow of membrane is actually taking place.

The alternative is that developing mastigonemes detach from their membrane associations and move, free in the cytoplasm. 67 to a new site where the next stage in their synthesis occurs.

This is intuitively unlikely and mastigonemes, free in the cytoplasm, without membrane associations have never been reported. In addition, from the kinetic studies of

mastigoneme development reported here, it can be seen that the transport of mastigonemes, along with membrane from the

perinuclear space to the Golgi apparatus can be detected with­

in two minutes after deflagellation of the organism (Figure

45). Likewise it can be seen that after mastigonemes have

accumulated in the Golgi apparatus after deflagellation in

colchicine, they are being depleted from the Golgi apparatus

within ten minutes after release from colchicine inhibition

(Figure 46). This suggests that the dynamic flow of membrane

through the cell is not a sluggish phenomenon.

Control of mastigoneme arrangement

The presence of mastigoneme organizational asymmetry on the long flagellum (a row of "tufts" of 2-4 on one side

vs. a row of single mastigonemes on the other side) invites

careful analysis of the developmental stages to try to deter­

mine at what level(s) this asymmetry is introduced. If

mastigoneme assembly is initiated by a site on the outer

nuclear membrane, then the specialization or spacial

asymmetry of these sites may to some extent control the final

asymmetric distribution of mastigonemes on the flagellum. 68

Figures 8 and 10 suggest that PMs may develop in two groups in two separate dilations of the perinuclear space; this may represent a special separation of PMs destined to be in

"tufts" from those to be found as singles, although no convincing evidence for this could be found. The transfer of PMs from the PNS to the cell surface would have to be quite precise with tufts and singles being released at those particular places that would allow them to be carried to their correct destination on the new flagellum as membrane is "pulled" up by the growing flagellum. This level of control is presumably accomplished by the Golgi apparatus.

The segregation of "tufts" from singles in the Golgi is strongly suggested by Figure 36 where groups of 2-3 PMs can

be seen in the dilations at one end and single PMs in dila­

tions at the other end. Thus morphology suggests at least

tentatively that the final arrangement of mastigonemes is

established by a fairly precise cytoskeletal linkage be­

tween nucleus and flagellar surface in which the "tufts" are

segregated from the single PMs and directed to opposite

sides of the flagellum. This scheme is diagramatically

illustrated in Figure 47.

Nuclear envelope-perinuclear space function

The nuclear envelope appears to be more than just a

boundary separating nucleus and cytoplasm. It has long been

recognized that the outer membrane of the nuclear envelope is 69 studded with ribosomes, making it structurally similar to the granular endoplasmic reticulum. This is confirmed by the similar biochemical make-up of nuclear envelope and micro­ somal cell fractions (33). The space between the inner and outer membranes of the nuclear envelope, the perinuclear space, is then functionally similar to the cisternae of the endoplasmic reticulum, and in many cases is continuous with it (33). This similarity is supported by the fact that PMs can be found developing in either the perinuclear space or cisternae of the endoplasmic reticulum (45, 47, 62).

In the organisms studied here, PMs develop in the perinuclear space and when large mature bundles are formed, they appear to pinch off from the nuclear envelope and become free vesicles in the cytoplasm. These vesicles look like dilated cisternae of endoplasmic reticulum packed with presumptive

mastigonemes. Early stages of assembly were never seen

in these cytoplasmic vesicles. This sequence suggests that

endoplasmic reticulum may arise from the nuclear envelope,

a suggestion that has been reported previously (33).

In addition to endoplasmic reticulum-like functions,

there have been reports of structures in the perinuclear space

indicating some other undefined functions. Membraneous tubu­

lar structures have been observed in the perinuclear space of oocytes of the African lungfish (99) and in crayfish spermato- 70 cytes (81). Proteinaceous crystalline inclusions have been re­ ported in the perinuclear space of leaves of several plant species (92, 109). In some cases these inclusions could be found in both the endoplasmic reticulum and the perinuclear space (109). The perinuclear space also has some affinity for symbiotic and parasitic organisms. It is the site of accumulation of some animal (91) and plant (2 7) viruses and a bacterial endosymbiont of Paramecium (54). The proximity of the perinuclear space to the genetic material of the cell may allow for ease of expression of some genetic information. The assembly of mastigonemes in the perinuclear space might allow the study of this information transfer. A scheme for the molecular assembly of mastigonemes can be visualized as follows. First, nuclea- tion or initiation sites are necessary on the inside of the outer nuclear membrane. These could be established by the binding of an informational macromolecule, from the nucleus, directly or indirectly via protein synthesis to the outer nuclear membrane. A rather precise positioning of these sites is required since the square-ended, antiparallel nature of the final assembly vesicle is determined at this point. Next, the synthesis of the protein components is needed; this most likely occurs on the ribosomes of the outer nuclear membrane, with the product being released into the peri­ nuclear space. This is interesting in that it suggests 71 that the m-RNA, which codes for mastigonemal protein, can dis­ criminate between perinuclear and other ribosomes, perhaps by virtue of their nuclear membrane associations. The subsequent development of mastigonemes from the protein subunits probably occurs by a self-assembly process as has been demonstrated

in many other protein subunit structures (47, 48, 56, 57),

in which the information required for the assembly of the

final structure is incorporated into the amino acid sequence of the subunits. Bouck (11) has reported that mastigonemes

have a single major protein subunit; however, it is possible

that others are present in undetectable amounts. For instance,

it seems reasonable that the tapered base and terminal fila­

ments may have a different subunit than the tubular shaft.

If this were the case, the base would be assembled first and

then act as a nucleation site for the assembly of the' shaft

from a second protein component. The shaft could in turn act

as a nucleation site for assembly of terminal filaments from

a third component. It appears that at least two orders of

information are required for the synthesis of mastigonemes

in the perinuclear space, information in the form of the amino

acid sequence of the protein subunit(s) and membrane mediated

information in the form of nucleation sites on the outer nuclear membrane. In summary, the nuclear envelope appears

to be a part of the endoplasmic reticulum with rather

specialized capabilities, perhaps by virtue of its proximity 72 to the nucleus.

It should be mentioned that a second site of membrane mediated information is required in the Golgi apparatus for the addition of carbohydrate moieties at the correct place on the mastigoneme. One may speculate that a combination of the appropriate spacing of functional groups on the mastigoneme, which is a function of the amino acid sequence of the subunits, and the appropriate spacial organization of enzymes on the Golgi membranes could accomplish this.

Williams (113) suggested such as an informational role for the Golgi apparatus in the assembly of coccoliths in Hymeno- monas as did Whaley et al. (112) for cell coats.

Kinetics of Mastigoneme Assembly and Transport

The nature of thin sectioning as a sampling technique must be considered in one's interpretation of the data shown

in Figures 45 and 46. The results are given as the per­

centage of a particular type of section; e.g., a mid-nuclear section containing nucleolus and plastid, which contained a

particular ultrastructural entity; e.g., PMs in the peri­

nuclear space. These percentages are only indirectly related

to the percentage of organisms in the sample which actually

possessed that entity. For example, if a perinuclear space

contains a bundle of eight mastigonemes, any single section 73 through that nucleus, depending on the plane of the section, may show; 1) no PMs, if the bundle is missed by the section;

2) all eight of the PMs, if the bundle is cross-sectioned; or 3) three or four PMs, if the bundle is cut in longi­ tudinal sections. Similarly, a change in the number of

Golgi apparatus sections which contain PMs may be due to either a change in the percentage of Golgi apparatus which contain PMs or in the number of PMs found in the average Golgi apparatus. Although general trends are convincingly

shown in Figures 45 and 46, for the above reasons stringent

comparisons and excess interpretation of the data are not

warranted and the quantitative calculations of mastigoneme

assembly, transport and utilization rates to be given below

are only first order approximations of the actual rates.

The results of these kinetic studies (Figures 45, 46)

support our interpretation of the process of reflagellation

and associated PM activity. When the cell is deflagellated,

the PMs move a few at a time from the large perinuclear bundles

into the Golgi apparatus, resulting in a decrease in the num­

ber of large bundles and an increase in both the number of

small bundles and the number of Golgi apparatus with PMs.

This response can be detected within minutes after deflagella- tion, before regeneration begins, indicating that the re­

quirement for a new flagellum, as opposed to flagellar growth,

is probably sufficient to stimulate PM transport to the Golgi 74 apparatus.

From the number of mastigonemes per ym of flagellum

(Figure 20) and the reflagellation kinetics curve (Figure 45,

Curve A) the rate at which mastigonemes are released to the flagellum can be calculated. From counts made on several micrographs similar to Figure 20 it has been determined that there are approximately 20 mastigonemes per ym of flagellum.

Curve Af Figure 45 shows that the maximum rate of flagellar regeneration is approximately 0.15 ym per minute. The calculation of the maximum rate of mastigoneme release is as follows: # of mastigonenes released per minute = # of mastigonemes

per ym of flagellum X # of ym of flagellar growth per

minute.

Therefore

# of mastigonemes released per minute = 20 mastigonemes

per ym of flagellum X 0.15 ym of flagellar growth per

minute = 3 mastigonemes per minute.

Therefore three mastigonemes are released per minute during

maximum flagellar growth.

As growth rate of the flagellum slows, fewer mastigonemes

per minute are required, and in response to this, the number

of Golgi apparatus sections with PMs begins to drop after 30

minutes, probably indicating that each Golgi apparatus con­ tains fewer PMs. Between 60 and 120 minutes after deflagella- 75 tion, the number of large perinuclear bundles of PMs begins to increase again, suggesting that the mastigoneme requirement of the slowly growing flagellum has fallen below the synthe­ sizing capability of the perinuclear space.

Large bundles of PMs are found in the perinuclear space of 0. danica (12) and O. minute during all stages of the cell cycle; therefore it appears that the assembly of PMs in this space is a relatively constant process in Ochromonas. As­ suming this to be true, the rate of PM assembly in the peri­ nuclear space can be calculated from the rate of PM utiliza­ tion at the point of inflection of Curve B in Figure 45 (% of perinuclear bundles of PMs with > 6 PMs). The rational for this assumption is as follows ; At the onset of reflagel­ lation, the rate of PM utilization is great, exceeding the rate of PM assembly in the perinuclear space and therefore the number of PMs per perinuclear bundle decreases. As the flagellar growth rate diminishes the rate of mastigoneme utilization likewise diminishes until it falls below the rate of PM assembly in the perinuclear space. At this point the number of PMs per bundle in the perinuclear space begins increasing. The point of inflection of Curve B in Figure

45 is at 60 minutes on the X-axis; at this point the flagel­

lum is growing at a rate of 0.06 ym per minute. If there are

20 mastigonemes on one ym of flagellum there are 1.2

mastigonemes required for 0.06 ym of new flagellum. Therefore 76 the rate of PM utilization at the point of inflection of Curve B in Figure 45 is 1.2 PMs per minute. For the rationale given above this can also be considered a first order approxi­ mation of the rate of PM assembly in the perinuclear space.

Figure 46 graphically shows the results of an experiment in which deflagellated cells were treated immediately with colchicine for one hour and then washed and transferred back into growth medium and allowed to reflagellate. While the simple deflagellation experiment (Figure 45) suggested that deflagellation alone would stimulate PM transport, colchicine and deflagellation treatment together allow one to distinguish

more clearly between the "need" for a new flagellum vs.

actual flagellar growth. Figure 46 shows that 15 minutes

after deflagellation in colchicine, there has been a transfer of PMs from the perinuclear space to the Golgi apparatus, even

though flagellar growth is inhibited. After 60 minutes in

colchicine, however, the percentages of PMs at the various loca­

tions have remained static suggesting that the lack of flagel­

lar growth has caused a "bottleneck" in the transport process.

Upon the removal of the inhibition and initiation of flagel­

lar growth, the response is seen in the process closest to the

"bottleneck;" i.e., in the release of accumulated PMs from

the Golgi apparatus to the newly growing flagellum. Shortly

thereafter the perinuclear PM bundles respond by channel­

ing more PMs into the Golgi apparatus. This is seen 77 particularly in sample 20pCh as the transformation of large bundles to small bundles and the corollary increase in the percentage of Golgi apparatus with PMs. The remainder of the process is similar to the simple reflagellation case shown in Figure 45.

Bouck (12) has suggested that the lag period between deflagellation and the appearance of a flagellar stub may be due to the time requirement of processing PMs through the

Golgi apparatus. In this regard it is significant that as organisms that have been deflagellated and treated in colchi­ cine accumulate PMs in their Golgi apparatus and when they are washed back into growth media, flagellar growth commences without a lag period. In O. danica the normal lag period is 15-20 minutes (12, 98) indicating that the processing of

PMs through the Golgi apparatus requires approximately 15-20 minutes. In O. minute the normal lag period is only 7-10 minutes (Figure 45). This difference is probably due to the smaller size of O. minute resulting in a shorter path for

PMs between the perinuclear space and cell surface. The fact that within 15 minutes after deflagellation in colchicine the % of Golgi apparati with PMs has reached a level that remains constant throughout the colchicine treatment (Curve

D, Figure 46) suggests that the time requirement of Golgi apparatus processing of PMs is less than 15 minutes.

These kinetic studies support the developmental se- 78 quence proposed by Bouck (11) as confirmed and expanded by us, and are more compelling than single static micrographs in defining the activity of the cell during the dynamic process of mastigoneme assembly and transport. 79

CONCLUSION

The intracellular assembly scheme that has been proposed for the mastigonemes of ochromonad flagellates (12) has been verified and elaborated on in this study. The kinetic data derived from reflagellating organisms offer a new approach to the study of mastigoneme assembly and transport with the potential of determining the time requirements for the assembly process. Also, the negative staining of osmotically disrupted organisms has allowed the study of the structure of whole pre­ sumptive mastigonemes in the perinuclear space with the reso­ lution afforded by the negative staining technique. Perhaps, more importantly, the potential of this system for the study of basic cell biological problems, including Golgi apparatus function, the dynamic flow of membrane through the membraneous organelles and the translation of genetic information above the self-assembly level, has been pointed out and discussed.

The advantages of this system for such studies include the manipulative potential of cells in culture; i.e., the ease of control of the chemical and nutritional environment, the manipulation of flagellar growth offered by deflagellation and colchicine inhibition, the ease of isolation of mastigonemes in quantities sufficient for biochemical anal­ ysis (12) and the statistical advantage of small cells for thin section electron microscopic analysis. 80

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ACKNOWLEDGMENTS

I wish to express my deepest appreciation to Dr.

Darryll E. Outka, my major professor, teacher, counselor and friend for his suggestions and assistance throughout the course of this work. Without his encouragement, wise counsel and unselfish patience this dissertation would not have been written.

I would also like to thank my Ph.D. committee members and fellow graduate students in the microscopy laboratory for their invaluable discussions of this work and professional and personal advice.

I am especially gratëful to my wife, Linda, for her encouragement, sacrifice and love throughout the years of this work and to my children, Eric and Lisa, for the joy they have brought me and for putting up with a dad that works long hours. This study was supported in part by a Dissertation

Fellowship from the Graduate College, Iowa State University. 92

APPENDIX A

Cerophyl Infusion Medium

Component Amount Final Concentration

A. Cerophyl extract ( 5%) 10-40 ml 0.05-0.2% B. Osterhaut solution 15 ml 1.5% C. KH2PO4 (0.05M) 3 ml D. Na2HP04 (0.05M) 7 ml }0.5 mM E. Distilled HgO q.s. to 1000 ml

Osterhaut Solution:

Component Amount

NaCl 0.105 g MgCl? 0.0085 g KCl 0.0023 g MgSO. 0.0040 g Cad. 0.0010 g HgO q.s. to 1000 ml Osterhaut solution can be made at 100 times these concentra­ tions and diluted.

Preparation of 5% Cerophyl extract:

Cerophyl is a dried grass preparation obtained from Cerophyl Labs, Kansas City, Mo. For each 100 ml of extract, disperse 5 g Cerophyl powder in 100 ml distilled water, heat 10 minutes at simmering boil, cool, filter through Whatman #1 filter paper and restore to volume. The extract can be tubed in 10 ml volumes and autoclaved at 15 p.s.i. for 15 minutes. The product can be refrigerated indefinitely.

Assembly of the media;

Components B through E are combined, dispensed in 500 ml volumes in one litre flasks, and autoclaved at 15 p.s.i. for 15 minutes. The final pH should be 7.2; this can be varied by changing the ratio of components C and D. The solution can be stored in the sterile state at room temperature until needed. At the time of use 5-20 ml of the 5% Cerophyl ex­ tract is added aseptically to give the desired Cerophyl con­ centration. 93

APPENDIX B

Method of Data Collection and Raw Data for Figures 45 and 46

The data presented graphically in Figures 45 and 46 were collected in such a way as to minimize observer bias. Three or four grids full of sections were cut from each sample block and stained. These grids were then placed in an LKB grid box (LKB INSTRUMENTS, INC., Rockville, Maryland, Catalog # 4828B) in random order and the position of each grid recorded. Two days later the grids were examined and the data recorded without knowledge of the sample source of the grids. The data were then compiled according to the sample from which they came. The raw data collected and calculated per­ centages are shown in the table on the following page. Table 1. Raw data for Figures 45 and 45 MID-NUCLEAR SECTIONS Sample Total # with % # with % # with % # >6 of 0 of <6 of ex amined P Ms/PNS total PMs/PNS total PMs/PNS total

Control 136 70 51*'b 42 31 24 18*'^ 2 min. post 160 68 43^ 66 deflagellation 41 26 16^ 15. min post 146 52 36^ deflagellation 44 30 50 34^ 30 min. post 101 25 25^ 39 deflagellation 39 37 37^ 60 min. post 117 21 18^ deflagellation 61 52 35 30^ 120 min. post 148 44 30^ deflagellation 62 42 42 28^ 15 min. in 129 52 41^ colchicine 41 31 36 28^ 60 min. in 136 48 35^ 56 41 32 24*^ colchicine 10 min. post release from 101 36 35 40 40 25 25^ colchicine 20 min. post release from 125 35 28^ 46 37 44 35^ colchicine 45 min. post 138 28 20^ release from 66 48 44 32^ colchicine 100 min. post release from 120 35 29^ 52 44 33 27^ colchicine

Points plotted on Figure 45.

Points plotted on Figure 46. 95

GOLGI APPARATUS SECTIONS Total # % # % # with of without of examined PMs total PMs total 74 10 13^'b 64 87

86 20 23^ 66 77

120 58 48^ 62 52

85 47 55^ 38 44

95 31 33^ 64 67

144 30 21^ 114 80

83 24 29^ 59 71

64 15 23^ 49 77

84 10 12^ 74 88

69 22 32^ 47 68

68 16 24^ 52 77

76 19 25^ 57 75