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THE ULTRASTRUCTURE OF PHYSARUM POLYCEPHALUM
USING CRYOe-TECHNIQUES

presented by

Monica Louise Converse Czymmek

has been accepted towards fulﬁllment
of the requirements for

MS Botany and Plant

degree in

 

 

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THE ULTRASTRUCTURE OF PHYSARUM POLYCEPHALUM
USING CRYO-TECHNIQUES
By

Monica Louise Converse Czymmek

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1996

ABSTRACT

THE ULTRASTRUCTURE OF PHYSARUM POLYCEPHALUM
USING CRYO-TECHNIQUES

By

Monica Louise Converse Czymmek

The ultrastructure of Physarum polycephalum was examined using cryo-
techniques. Plasmodia were studied using low-temperature scanning electron
microscopy and high-pressure freezing and freeze-substitution with transmission
electron microscopy. Low-temperature scanning electron microscopy allowed
observations of the slime layer deposited by the migrating plasmodia, plasmodial
veins, and slime filaments. Plasmodia examined by transmission electron
microscopy contained microfibrils adjacent to the smooth-contoured plasma
membrane and throughout the cytoplasm. Numerous vacuoles were seen of variable
shape, size, and content. Nuclei, globular mitochondria, golgi, contractile vacuoles,
food vacuoles, and many membrane bound vesicles of unknown composition also
were present. Overall preservation of plasmodial membranes and other cytoplasmic
structures was excellent, with minimal ice crystal damage. Observations of
sporangia using low-temperature scanning electron microscopy revealed a double-
walled peridium with small openings at its surface and ornamented sporangiospores,
each containing a large depression. Overall, cryo-preservation provided more

accurate morphological details than conventional chemical fixation techniques.

ACKNOWLEDGMENTS

I thank Dr. Karen Klomparens for her patience and support during my
degree. I would also like to thank Connie Bricker and Laura Sadowski at Miami
University, Oxford, Ohio for assistance in the use of the Balzer's High Pressure
Freezer (National Science Foundation grant No. DIR 88-20387 to Dr. Martha Powell
and Dr. Allen Allenspach). I am grateful to Dr. Henry Aldrich at the University of
Florida at Gainesville for his generous donation of cultures of Physarum
polycephalum. I also thank Dr. Kirk J. Czymmek for his freeze-substitution
protocol as well as his encouragement and Austen Converse Czymmek for his

understanding during the course of my thesis.

iii

TABLE OF CONTENTS

LIST OF FIGURES ................................................................................................... vi

INTRODUCTION ...................................................................................................... 1

CHAPTER I. Ultrastructural observations of Physarum polycephalum

plasmodia using cryo-techniques ..................................................... 3
Abstract ...................................................................................................................... 3
Introduction ................................................................................................................ 4
Materials and Methods .............................................................................................. 5
Results ......................................................................................................................... 6
Discussion ................................................................................................................. 50

CHAPTER II. Low-temperature scanning electron microscopy of Physarum

polycephalum sporangia ................................................................. 57
Abstract .................................................................................................................... 57
Introduction .............................................................................................................. 58
Materials and Methods ............................................................................................ 59
Results ....................................................................................................................... 60
Discussion ................................................................................................................. 78
SUMMARY .............................................................................................................. 82
LIST OF REFERENCES ......................................................................................... 84

F i

Fi

LIST OF FIGURES

CHAPTER I

Figure 1 - Low magniﬁcation LTSEM of Physarum polycephalum plasmodium .. 10

Figure 2 - LTSEM of Physarum polycephalum plasmodial surface ........................ 12
Figure 3 - LTSEM of Physarum polycephalum plasmodial surface ........................ 12
Figure 4 - LTSEM of Physarum polycephalum plasmodial surface ........................ 12
Figure 5 - LTSEM of Plzysarum polycephalum plasmodial strands ........................ 14
Figure 6 - High magniﬁcation LTSEM of plasmodial strands ............................... 16
Figure 7 - High magniﬁcation LTSEM of plasmodial strands ............................... 16
Figure 8 - LTSEM of Physarum polycephalum plasmodial coalescence ................. 18
Figure 9 - LTSEM of Physarum polycephalum plasmodial coalescence ................. 18
Figure 10 - LTSEM of Physarumpolycephalum slime ﬁlaments ............................ 20
Figure 11 - LTSEM of Physarumpolycephalum slime'ﬁlaments ............................ 20
Figure 12 - LTSEM of Physarum polycephalum slime ﬁlaments ............................ 20
Figure 13 - Glutaraldehyde-ﬁxed LTSEM of P. polycephalum plasmodium ......... 22
Figure 14 - Glutaraldehyde-ﬁxed LTSEM of P. polycephalum plasmodium ......... 22
Figure 15 - Glutaraldehyde—ﬁxed LTSEM of P. polycephalum plasmodium ......... 22
Figure 16 - Cryo-fractured LTSEM of Physarum polycephalum plasmodia .......... 24
Figure 17 - Cryo-fractured LTSEM of Physarum polycephalum plasmodia .......... 24
Figure 18 - High magniﬁcation of cryo-fractured plasmodia ................................. 26
Figure 19 - High magniﬁcation of cryo-fractured plasmodia ................................. 26

Figure 20 - HPF of Physarum polycephalum plasmodial invaginations .................. 28

 

Figure 21 - HPF of Physarumpolycephalum plasmodial invaginations .................. 28

Figure 22 - HPF of Physarum polycephalum plasmodial channels ......................... 30
Figure 23 - HPF of Physarum polycephalum contractile vacuoles .......................... 32
Figure 24 - HPF of Physarum polycephalum contractile vacuoles .......................... 32
Figure 25 - HPF of Physarum polycephalum invaginations .................................... 34
Figure 26 - HPF of Physarum polycephalum invaginations .................................... 34
Figure 27 - HPF of Physarum polycephalum cytoplasm .......................................... 36
Figure 28 - HPF of Physarum polyceplmlum cytoplasm .......................................... 36
Figure 29 - HPF of Physarumpolycephalum mitochondria .................................... 38
Figure 30 - HPF of Physarum polycephalum mitochondria ................................... 38
Figure 31 - HPF of Physarum polycephalum nucleus .............................................. 40
Figure 32 - H PF of Physarum polycephalum food vacuole ...................................... 42
Figure 33 - HPF of Physarum polycephalum vacuoles ............................................. 44
Figure 34 - HPF of Physarum polycephalum vacuoles ............................................. 44
Figure 35 - HPF of Physarum polycephalum vacuoles ............................................. 44
Figure 36 - HPF of Physarum polycephalum slime ﬁlaments .................................. 46
Figure 37 - HPF of Physarum polycephalum slime ﬁlaments .................................. 46

Figure 38 - HPF of Physarum polycephalum slime ﬁlaments & calcium deposits.. 48

Figure 39 - HPF of Physarum polycephalum slime ﬁlaments & calcium deposits.. 48

vi

CHAPTER [I

Figure 40 - LTSEM of Physarum polycephalum sporangial initials ....................... 62
Figure 41 - LTSEM of Physarum polycephalum sporangia .................................... 64
Figure 42 - LTSEM of Physarum polycephalum sporangia .................................... 64
Figure 43 - LTSEM of Physarum polycephalum sporangia .................................... 66
Figure 44 - LTSEM of Physarum polycephalum sporangia .................................... 66
Figure 45 - LTSEM of Physarum polycephalum mature sporangia ........................ 68
Figure 46 - LTSEM of Physarum polycephalum peridial surface ........................... 70
Figure 47 - LTSEM of Physarum polycephalum peridial surface ........................... 70
Figure 48 - LTSEM of Physarum polycephalum peridial surface ........................... 70
Figure 49 - Cryo-fractured LTSEM of Physarum polycephalum sporangia .......... 72
Figure 50 - Cryo-fractured LTSEM of Physarum polycephalum sporangia .......... 72
Figure 51 - LTSEM of Physarum polycephalum spores .......................................... 74
Figure 52 - LTSEM of Physarum polycephalum spores .......................................... 74
Figure 53 - LTSEM of Physarum polycephalum spores and capillitia .................... 76

Figure 54 - LTSEM of Physarum polycephalum spores and capillitia .................... 76

vii

INTRODUCTION

Physarum polycephalum, a member of the Myxomycetes, is characterized by
a plasmodial vegetative stage when exposed to continuous darkness and a
sporophore stage leading to the production of haploid spores under starvation and
continuous light. The phaneroplasmodium of P. polycephalum consists of a
protoplast surrounded by a plasma membrane devoid ofa cell wall. The
plasmodium exhibits protoplasmic streaming up to 1mm/s with a reversible
streaming period of 1.5 to 3 minutes (Sauer, 1982). A slime layer or glycocalyx
surrounds the plasmodium and extends into plasmalemmal invaginations, serving as
a protective layer against desiccation and external ionic ﬂuctuations (Wolf et al.,
1981a; and Kessler, 1982).

Many aspects of the life cycle of P. polycephalum have been documented
biochemically and genetically. Sauer (1982) noted that many of the biochemical
studies of the contractile proteins actin and myosin utilized P. polycephalum as the
specimen of choice due to its ease of culture and its large size. However,
ultrastructural studies to date have been of poor quality and/or incomplete due to
ﬁxation difﬁculties as a result of the high water content of many structures. With
recent technological advances, many of these problems can be overcome.

In low-temperature scanning electron microscopy, plunge freezing rapidly
immobilizes dynamic biological processes in milliseconds rather than minutes when

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using conventional chemical ﬁxation (Gilkey and Staehelin, 1986). Samples are
preserved with no exposure to chemical ﬁxatives or solvents (Beckett and Read,
1986). With high-pressure freezing, samples up to 600nm in diameter may be
successfully prepared (Gilkey and Staehelin, 1986) allowing more accurate
observations of plasmodial structures.

This research involves cryo-preparation of selected studies in the life cycle of
P. polycephalum. This includes low-temperature scanning electron microscopy and
high-pressure frozen freeze-substituted samples for transmission electron
microscopy of plasmodia and low-temperature scanning electron microscopy of
sporangia. The purpose of this research is to better deﬁne many aspects of the P.

polycephalum life cycle using the above mentioned cryotechniques.

 

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CHAPTER I

ULTRASTRUCTURAL OBSERVATIONS OF PHYSARUM POL YCEPHALUM

PLASMODIA USING CRYO-TECHNIQUES

ABSTRACT

The ultrastructure of Physarum polycephalum plasmodia was examined using
low-temperature scanning electron microscopy and high-pressure freezing with
freeze-substitution for transmission electron microscopy. Low-temperature
scanning electron microscopy revealed phaneroplasmodia consisted of a protoplast
with veins and an advancing front of slime ﬁlaments. The net-like plasmodial front
coalesced by the projection of plasmodial strands across the areas not encompassed
yet by the plasmodial body. High-pressure frozen freeze-substituted samples viewed
by transmission electron microscopy allowed observations of internal details of
plasmodia. Microﬁbrils were seen on the cytoplasmic side beneath the plasma
membrane and adjacent to the plasmalemmal invaginations. Globular
mitochondria with prominent rod-like nucleoids, single cisternae of golgi, rough
end0plasmic reticulum vesicles, food vacuoles, contractile vacuoles, and various
types of unclassiﬁed vesicles were observed in the plasmodia. High-pressure
freezing proved to be very effective in rapidly immobilizing biological events of

Physarum polycephalum with minimal ice crystal damage.

 

 

 

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INTRODUCTION

For many years the Myxomycetes were classiﬁed on the basis of sporophore
development and structure. Then in 1968, Gray and Alexopoulos characterized the
vegetative or plasmodial stage as having three distinct types: protoplasmodium,
aphaneroplasmodium, and phaneroplasmodium. Alexopoulos (1982) described a
fourth, unnamed type characteristic of the Order Trichiales which is intermediate
between the aphaneroplasmodium and the phaneroplasmodium but has not been
studied extensively. The protoplasmodium is microscopic with slow, irregular
protoplasmic streaming, there are no distinct veins or channels, and is characteristic
of the Order Echinosteliales and some members of the Liceales (Haskins and
Hinchee, 1974). The aphaneroplasmodium is produced by the Stemonitales and
exhibits ﬂattened, nongranular veins with irregular, rhythmic, reversible flow which
varies in speed from fast to unrecognizable. No ﬁbrous glycocalyx or slime sheath
has been demonstrated (Haskins and Hinchee, 1974).

Physarum polycephalum, as well as other members of the Physarales, forms
phaneroplasmodia consisting of anterior fleshy fans of protoplasm with thick,
tubular veins in which rhythmic, reversible flow is demonstrated. The
phaneroplasmodium is enveloped by a glycocalyx containing microﬁbrils
(Alexopoulos, 1982).

Physarum polycephalum has been the center of numerous genetic and
biochemical studies. Its plasmodial ultrastructure has been studied with

transmission electron microscopy (TEM) and scanning electron microscopy (SEM)

 

 

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using conventional chemical ﬁxation (Wohlfarth-Bottermann, 1962, 1963, 1964;
Rhea, 1966; Aldrich, 1967; Goodman and Rusch, 1970; Daniel and Jarlfors,
l972a,b; Chet and Kislev, 1973; Haskins and Hinchee, 1974; Lane and Carlile,
1979; Wolf and Stockem, 1979; Wolf et al. 1981a; Kessler, 1982; Salles-Passador et
al., 1991; and Kuroiwa, et al., 1994). Aldrich (1982) mentioned the difﬁculty in
studying myxamoebae, swarm cells, and plasmodia due to their high water content.
Likewise, many artifacts are introduced with TEM and SEM studies using
conventional chemical ﬁxation (Aldrich, 1982). Aldrich (1989) discussed various
problems associated with glutaraldehyde ﬁxation including shrinkage and that P.
polycephalum has been shown to continue streaming and changing for one minute
or more after application of 2.5% glutaraldehyde.

Recently, improvements in cryo-techniques, including low-temperature
scanning electron microscopy (LTSEM) and high-pressure frozen freeze-substituted
(HPF-F S) samples for TEM have allowed more accurate observations of specimens.
Plunge freezing samples of LTSEM rapidly immobilizes dynamic biological
processes in milliseconds rather than minutes when compared to conventional
chemical techniques, preserves samples without exposure to chemical ﬁxatives or
solvents, and eliminates dehydration artifacts (Beckett and Read, 1986).

This study examined plasmodial morphology and development using LTSEM
and HPF-FS samples for TEM which were inaccurate or unobtainable with
conventional chemical techniques. This research is essential for providing more

reliable morphological and developmental details in P. polycephalum.

 

 

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MATERIALS AND METHODS

Cultures of P. polycephalum (Courtesy of H. C. Aldrich) were maintained on
2% water agar with oat ﬂakes in continuous dark. Samples for LTSEM were grown
on 4mm agar squares in continuous dark and then mounted on a cryo-holder which
was covered by a thin layer of Tissue-Tek II (Lab-Tek Products, Division of Miles
Laboratories, Naperville, II 60540) with graphite, plunge frozen in a slurry ofliquid
nitrogen, etched at -65°C for 5-10 minutes, gold coated in an EMscope SP2000
Sputter—Cryo System, and then observed, while frozen-hydrated, at 10 kV with a
JEOL 35CF scanning electron microscope. Fractured samples were struck with a
cryo-knife immediately after being plunged in liquid nitrogen and then processed as
described above.

Chemically ﬁxed plasmodia for LTSEM were grown as described above,
ﬂooded with 4% glutaraldehyde in phosphate buffer (pH 7.2) at room temperature,
rinsed 2X in distilled water and processed for LTSEM as described above.

Plasmodia for TEM were grown in 1.5mm wide sterilized gold high-pressure
freezer hats on small pieces of 2% water agar for approximately 20h in continuous
dark prior to freezing. 20% dextran (MW 39,100) was added to the sample as a
cryoprotectant 5 minutes before freezing in a Balzer's HPM 010 high-pressure
freezer. Samples were transferred and stored in liquid nitrogen until they could by
processed further. Samples were freeze-substituted according to Czymmek and

Klomparens (1992) in a RMC M86200 holding device in a solution of 0.05% uranyl

 

 

 

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Samples were then brought to room temperature at 2h intervals at -35°C, -25°C, -
12°C, 0°C, 10°C, and room temperature. Specimens were removed from the high
pressure freezer hats, placed in glass vials, rinsed 3X in 100% acetone, and
inﬁltrated and embedded under desiccation (calcium sulfate and phosphorous
pentoxide) with a slight vacuum in Spurr's epoxy resin ( Spurr, 1969) in a graded
series of 10%, 25%, 40%, 50%, 75%, 85%, 95%, 100% with acetone as the solvent.
Ultrathin sections were stabilized on colloidion and carbon-coated grids, stained
with 0.5% uranyl acetate and Reynolds' lead citrate (Reynolds, 1963), and observed

with a JEOL 100CX 1] transmission electron microscope.

RESULTS
Low-temperature scanning electron microscopy plasmodia

Plasmodia prepared for LTSEM illustrated signiﬁcant improvements over
previous chemically ﬁxed studies mentioned earlier. The LTSEM studies revealed a
well preserved plasmodia that consisted of thick posterior veins and an advancing
front of protoplasm (Fig. 1). The plasmodial front (Fig. 2) consisted ofa net-like
appearance with small openings representing regions not yet encompassed by the
plasmodium. The well preserved extracellular slime layer or glycocalyx was seen on
the surface of the agar surrounding the plasmodium. Figures 1—4 illustrate the
various growth patterns exhibited by P. polycephalum. The net-like appearance

(Figs. 1 and 2) was the most common type observed.

 

The net-like plasmodial front later coalesced by the projection and expansion
of plasmodial strands across the areas not encompassed yet by the plasmodial body.
The plasmodial strands frequently appeared to emanate from different levels
resulting in a tiered appearance (Fig. 5). Figures 6-9 illustrate the plasmodial
strands extending across open areas of the plasmodium. Many areas were not ﬁlled
in completely by the strands and remained as openings in the plasmodial surface.

The LTSEM studies revealed a well preserved plasmodia that consisted of
slime ﬁlaments that extended down from the sides of the plasmodium (Figs. 10-12).

Glutaraldehyde-ﬁxed samples showed numerous plasma membrane
invaginations (Figs. 13-15) which was presumably an artifact of conventional
chemical ﬁxation perhaps caused by the plasma membrane invaginating to a much
greater degree than would occur under normal living conditions. For many years it
has been assumed by many researchers that the plasmodium consists ofa gelliﬁed
ectoplasm that is distinctly different than the fluid endoplasm. However, this
research showed no evidence of a gelliﬁed ectoplasm in LTSEM samples.

The LTSEM cryo-fractured samples allowed observation of internal details
of the plasmodia. Plasmodial structures were primarily smooth on the surface with
veins, small openings, and external slime ﬁlaments (Figs. 16-18). Internal details
revealed some ice crystal damage, as expected, and was identiﬁed as rough,
distorted areas in the cytoplasm (Fig. 19).

High-pressure frozen freeze-substituted plasmodia

 

 

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High pressure freezing in conjunction with freeze-substitution allowed
excellent preservation of the plasmodia and associated structures of P.
polycephalum. Mechanical damage due to the manipulation of the plasmodia was
minimized prior to high pressure freezing by growing the cultures on small pieces of
water agar inside the high pressure freezer gold hats for approximately 20 hours.
Plasmodia were preserved well with open penetrating channels that presumably had
not been encompassed by the plasmodial body (Fig. 20). Projections of the
plasmodia extended into the channels which contained vacuoles with variable
contents(Figs. 21 and 22). The protoplasm appeared very uniform with well
preserved organelles.

Vacuoles were observed (Figs. 23 and 24) with microvillus projections
extending inward from the cytoplasm, which presumably represent contractile
vacuoles. Rough endoplasmic reticulum (ER) vesicles frequently were observed
associated with the contractile vacuoles.

Plasmalemmal invaginations (Fig. 25) were observed with microﬁbrils,
presumably actin and myosin, parallel to the plasma membrane and adjacent to the
invaginations on the cytoplasmic side. Projections were seen extending into areas
not ﬁlled in by the protoplasm (Fig. 26). The plasmodial slime (glycocalyx) ﬁlled the
plasmalemmal invaginations. Figure 26 also illustrated the numerous rough ER
vesicles that appeared as small spherical vesicles with ribosomes associated to the

surface.

II)

The plasmodial cytoplasm (Figs. 27 and 28) typically contained many
different types of vacuoles of variable size. Some vacuoles had electron transparent
contents while others appeared as vesicles within vesicles. The golgi were observed
as single, oblong, flattened vesicles. Many golgi contained coated vesicles at one
end.

The HPF-FS plasmodia for TEM contained spherical mitochondria with
prominent DNA containing, electron dense nucleoids and tubular cristae (Figs. 29-
31) not in the form of lamellae typically observed in eukaryotes. Calcium inclusions
often were observed within the mitochondria.

Nuclei (Fig. 31) were preserved very well with high pressure freezing. Nuclei
were round in proﬁle with very regular nuclear envelopes. Chromatin was very
uniformly distributed and non-chromatin areas of the nucleoplasm were not leached
as seen previously with conventional chemical ﬁxation. Each interphase nucleus
contained a large, central nucleolus. An artifact of the nuclear envelope was
observed as a separation of the perinuclear space, presumably a result of resin
inﬁltration.

Many food vacuoles (Figs. 31, 32, and 35) of variable size and shape were
observed in the plasmodia. Blebs or fusion events were seen at the vacuolar surface
and ﬁbrillar material and debris were located within the food vacuole. Food
vacuoles prepared by chemical fixation do not have the ﬁbrillar material as observed

with HPF-F S samples for TEM. Instead, chemically ﬁxed food vacuoles typically

contained debris in an otherwise empty vacuole in which the membrane was very
disrupted.

Calcium deposits accumulated in speciﬁc vacuoles throughout the cytoplasm
(Figs. 33, 34, and 39). Calcium has been identiﬁed using x-ray spectroscopic
analysis in previous studies. It has been suggested that calcium concentration in the
cyt0plasm may be involved in the mechanism responsible for controlling
protoplasmic streaming in P. polycephalum.

Well preserved slime ﬁlaments (Figs. 36-38) were observed projecting from
the plasma membrane, as seen previously in the LTSEM samples (Figs. 10-12)in this
study. The slime ﬁlaments projecting from the plasma membrane were in direct

contact with the plasmodial slime layer.

FIG. 1. Low magniﬁcation LTSEM of Physarum polycephalum plasmodium.
LTSEM enabled more accurate observations of plasmodial morphology including
the smooth-contoured plasmodial surface, plasmodial front (F) and many veins (V).

Bar = 1000 um.

 

 

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FIGS 2-4. LTSEM of Physarum polycephalum plasmodial surface. Fig. 2.
Higher magniﬁcation of Fig. 1 shows the net-like plasmodial front (F) with areas
not yet encompassed by the plasmodial body (arrows). Bar = 500 um. Figs. 3 and 4.
Micrographs of the variable growth types of plasmodia; compare with Fig. 1. Bars

for Figs. 3 and 4 = 1000 um.

 

 

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FIG. 5. LTSEM of Physarum polycephalum plasmodial strands. Cryo-
preservation of samples revealed that many plasmodial strands (S) emanated from
different levels resulting in a tiered appearance that presumably will coalesce to
form the plasmodial body. Note the relatively smooth plasmodial surface

(arrowheads) with LTSEM preservation. Bar = 50 um.

 

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FIGS. 6-7. High magniﬁcation LTSEM of plasmodial strands. Various
stages in plasmodial development. Strands (arrows) emanated across areas not yet

encompassed by the plasmodia. Bars = 5 um & 20 um respectively.

 

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FIGS. 8-9. LTSEM of Physarum polycephalum plasmodial coalescence. The

strands (arrows) presumably coalesced to form the plasmodial body. Bars = 10 um.

 

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FIGS. 10-12. LTSEM of Physarum polycephalum slime ﬁlaments. Fig 10.
LTSEM samples revealed a smooth plasmodial surface (arrowheads) with numerous
slime ﬁlaments (F) that extended from its side. Bar : 50 um. Fig. 11. High
magniﬁcation of Fig. 10 showed the slime ﬁlaments (F) attached to the plasmodium.
Note the many openings (arrows) on the lower side of the plasmodium. Bar = 30

um. Fig. 12. Slime ﬁlaments (F) projected from the plasmodial front. The lower

side of the plasmodium showed many small openings (arrows). Bar = 50 um.

 

 

FIGS. 13-15. Glutaraldehyde-ﬁxed LTSEM of Physarumpolycepltalum
plasmodium. Fig. 13. Glutaraldehyde-ﬁxed sample was observed with LTSEM and
revealed extensive artifactual plasma membrane invaginations of the plasmodial
surface. In particular, plasmodial veins were covered with numerous invaginations
(arrows) of the plasma membrane which appeared as infoldings on the surface.

Note the absence of slime ﬁlaments. Bar = 100 um. Figs. 14 and 15. High
magniﬁcation of extensive plasma membrane invaginations on the plasmodial vein
surface (arrows) due to glutaraldehyde ﬁxation. Bars = 50 um and 20 um

respectively.

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FIGS. 16-17. Cryo-fractured LTSEM of Physarum polycephalum plasmodia-
Fig. 16. Cryo—fractured sample showed that slime filaments (F) extended down from
the plasmodium. Note the relatively smooth plasmodial surface (arrowhead). Bar =
50 um. Fig. 17. Cryo-fractured plasmodium with nucleus (N) and slime ﬁlaments
(F). Note the well preserved, smooth plasmodial surface (arrowheads) and the

absence of any distinct endoplasm and ectoplasm. Bar = 50 um.

27

I

1.1.. .
a) .

:W»...

 

. V
A

w
a...

r ,
2AM. .

28

FIGS. 18-19. High magniﬁcation of cryo-fractured plasmodia. Fig. 18.
Cryo-fractured plasmodium revealed internal details. With untreated LTSEM
samples, no regions were observed to indicate the presence ofa distinct separate
endoplasm and ectoplasm. Note the slime ﬁlaments (F) and nuclei (N). Bar = 20
um. Fig 19. High magniﬁcation of Fig. 18. Note the nuclei (N) and other

cytoplasmic material. Bar = 10 um.

 

 

29

.th NIH-VJ

x...
x ..

ox

..._,\

1 ,
..v y e h

, I
r

r:

1
II
.. "1 it.
. ﬁ
1

..

 

30

FIGS. 20-21. HPF of Physarum polycephalum plasmodial invaginations.
Low magniﬁcations of high pressure frozen freeze-substituted plasmodium with

nucleus (N), spherical mitochondria (M), and invaginations (I). Bars = 2 um and 1

pm respectively.

 

 

. 3:. I :9

vi",

.1;
’ ‘i

 

'r
a

 

'n f
tint": *

::€'Z'-' ’.

y
‘n

36.53 '
4 Q

“d
t?
»r

‘41 Ar:
$1.
1*

.c

an“

geisha-6135.111. «A1;

 

FIG. 22. HPF of Physarum polyceplmlum plasmodial channels. High
magniﬁcation of projections (P) within a channel. Note mitochondria (M) and

round rough endoplasmic reticulum (ER) vesicles. Bar = 1 pm.

 

34

FIGS. 23-24. HPF of Physurumpub’ceplmlum contractile vacuoles.
Presumptive contractile vacuoles contained microvilli (arrowheads) projecting from

the cytoplasm. Bars = 2 pm.

- we
,“r ‘n’9‘l
.. ~.

,)

"ewes-33¢ .
: we

I 31‘“
.. .
.3: ~"

0
..

 

36

FIGS. 25-26. HPF of Physarumpolyceplmlum invaginations. Fig. 25.
Invagination near the plasmodial surface which contained slime material (S) and a
large vesicle (V) with numerous small vesicles associated with its surface. Note the
smaller invaginations (arrowheads), plasma membrane (P), and ﬁbrillar areas
(arrows). Bar = 2 pm. Fig. 26. Invagination (I) containing cytoplasmic projections

(P). Note the round rough endoplasmic reticulum (ER) vesicles. Bar = l um.

 

 

 

38

FIGS. 27-28. HPF of Physarum polyceplmlum cytoplasm. Fig. 27. Note the
variety of vesicles within the plasmodium: mottled vesicles (mv), ﬁbrillar vesicles (1),
vesicles within vesicles (arrowhead), and golgi (G). Also note spherical
mitochondria (M) with prominent nucleoids (n). Bar = 1 pm. Fig. 28. Plasmodium
contained numerous vacuoles (V) and golgi (G) consisting of single cisternae. Note

the coated vesicle still attached to the golgi (arrow). Bar = 1 pm.

.P

' 5‘3“

. . .391 '
- >c..' v‘

tam-'WW' '
- . -./. A-

4 .' a! .- r? I u<

t

 

40

FIGS. 29-30. HPF of Physarum [mlyceplmlum mitochondria. Fig. 29.
Plasmodium nucleus (N) with a round proﬁle. Note the nucleolus (NU) and
mitochondria (M). Bar = 1 pm. Fig. 30. Note mitochondria (M) with tubular

cristae are round in proﬁle with prominent rod-like nucleoids (n). Bar = 1 pm.

 

 

 

is.
.

 

        

. (b...
v «r

2.
..n .18
Pt. .5

, - f
. 2.».

.1. \\.
.

   

u .

FIG. 31. HPF of Physarum [mb'ceplmlum nucleus. Well preserved nucleus
(N) with a single electron-dense nucleolus (NU) and hetero-chromatin (arrowhead).

Note mitochondria with regular tubular cristae (arrow) and food vacuole (FV). Bar

=1um.

 

 

44

FIG. 32. HPF of Physarum polyceplmlmn food vacuole. Food vacuole (F V)
contained partially digested cellular debris. Note the vesicles (arrowheads)
attached to the food vacuole membrane and the well preserved ﬁbrillar background

within the food vacuole. Bar = 0.5 pm.

 

40

FIGS 33-35. HPF of Physurmn [mlyceplmlum vacuoles. Figs. 33 and 34.
Vacuoles (V) with several electron dense regions (presumably calcium inclusions).
Also note the vesicular blebbing or fusion events (arrowheads) at the vacuolar
surface. Bars = 1 um. Fig. 35. A large food vacuole (FV) containing membranous
structures and numerous blebbing or fusion events (arrowheads) at its surface. Note

tubular cristae (arrow) in mitochondria (M). Bar = 1 pm.

47

“hymn ARK-Nd. .
I . . A , .
. AAA 32].“ .

0

.. a.

:JII.

4A..

 

48

FIGS. 36-37. HPF of Pltysarum polyceplmlum slime ﬁlaments. High
magniﬁcation of slime ﬁlaments (F) at the plasmodial surface. Note the

invaginations (arrows). Bars = 1 pm.

I .

"frl ' V.
. '1‘?

 

50

FIGS. 38-39. HPF of Pltys‘urum pulyceplmlmn slime ﬁlaments and calcium
deposits. Fig. 38. Many slime ﬁlaments (F) along the plasma membrane
surrounded by a relatively electron dense material which was presumably the
glycocalyx. Bar = 1 pm. Fig. 39. Electron dense regions (arrows) throughout the

cytoplasm probably represent calcium. Bar = 0.5 pm.

 

Leif]

.1.

 

m '4’“ ‘ C . ~. -
,f’n ,- L"; p ‘
3» ’"7 -.\-r , a“

24;.

‘n

v \.
4.

DISCUSSION

Plasmodia are particularly susceptible to membrane and dehydration
artifacts due to the absence ofa rigid cell wall and high water content. Myxomycete
plasmodial ultrastructure has been studied previously using convention chemical
ﬁxation techniques (Wohlfarth-Bottermann, l962,1963,1964; McManus, 1965;
McManus and Roth, 1965; Rhea, 1966; Aldrich, 1967; McManus and Roth, 1967;
Goodman and Rusch, 1970; Daniel and Jarlfors, l972a,b; Charvat et al., 1973; Chet
and Kislev, 1973; Haskins and Hinchee, I974; Lane and Carlile, 1979; Wolf and
Stockem, 1979; Wolf et al., 1981a; Kessler, 1982; Salles-Passador et al., 1991; and
Kuroiwa et al., 1994). However, much of these data are difﬁcult to interpret
ultrastructurally due to substantial altering of membranes and associated structures
with conventional ﬁxation techniques.

Gilkey and Staehelin (1986) noted that to have an effective ﬁxation protocol,
the ﬁxative must penetrate and act quickly to immobilize the specimen and crosslink
molecules, it should immobilize all the atoms and molecules in the cell, and it should
not alter the morphology of the organism. Aldrich (personal communication)
observed that plasmodia ﬁxed with 2.5% glutaraldehyde can continue to stream for
up to one minute or more. Gilkey and Staehelin (1986) cited several other studies in
which chemically ﬁxed cells have been known to continue streaming and changing
for several seconds to minutes following exposure to chemical ﬁxatives and noted

that chemical ﬁxation has been known to cause swelling or shrinkage of cells and

53

organelles, which are exhibited in ﬁxed material as wavy membranes due to osmotic
stress.

The use of LTSEM and high-pressure freezing with freeze-substitution
eliminated many problems associated with conventional chemical ﬁxation by rapidly
immobilizing membranes and other cellular structures. The LTSEM is a technique
described by Beckett and Read (1986) which allows the specimen to be rapidly
frozen in a slurry ofliquid nitrogen at a rate of 50 msec for a 150 um sample and
then observed in the frozen-hydrated state in the SEM, thus avoiding the
introduction of chemicals normally used for ﬁxation and dehydration. This
procedure provides excellent spatial preservation of membranes and their associated
cytoplasmic structures. Freezing in liquid nitrogen for LTSEM is not as rapid as
other ultrarapid freezing techniques such as high-pressure freezing, but it is
adequate for general morphological studies at the SEM level.

High-pressure freezing in conjunction with freeze-substitution was used in
this study to observe samples of P. polycephalum at the TEM level. Gilkey and
Staehelin (1986) discussed that high-pressure freezing can be used for samples up to
a thickness of 600 pm at a rate close to 100°K/sec, which is ideal for the relatively
large size ofP. polycephalum plasmodia. Ultra-rapid freezing methods such as high-
pressure freezing can ﬁx biological specimens orders of magnitude faster than
chemical ﬁxation and maintain spatial relationships among molecules and structures
(Gilkey and Staehelin, 1986). High-pressure freezing can utilize larger samples than

many other freezing techniques by subjecting the samples to high-pressure (2100

bars) before exposing them to the cryogen liquid nitrogen, thus slowing the rate of
ice crystal growth.

Many of the chemical ﬁxation studies mentioned earlier, as well as studies
with freeze-fracture techniques (Wolf et al., 1980) and high-pressure freezing (Wolf
et al., 1981b), underwent extensive manipulation, thus disruption of the plasmodia
during preparation ofthe specimen prior to ﬁxation. Many ofthese samples were
excised pieces of plasmodia, while others were protoplasmic drops obtained by
puncturing the plasmodial strands with a needle. Trauma-induced changes in
cellular structures may occur prior to freezing during specimen preparation
procedures and ultrarapid freezing techniques can only preserve cells in the
condition they were in before freezing, not before they were prepared for freezing
(Gilkey and Staehelin, 1986).

To avoid specimen preparation artifacts in this study, care was taken to a
refrain from manipulation of samples prior to high-pressure freezing. Plasmodia
were grown on small pieces of water agar inside the HPF gold hats for
approximately 20 hours in continuous dark. Dextran was then added as a
cryoprotectant and the samples were allowed to incubate for 5 minutes before being
placed in the high-pressure freezer. Dextran was chosen as the cryoprotectant due
to it being a nonpermeable cryoprotectant unlike the commonly used glycerol which
has been shown to cause osmotic disruption of membranes (Gilkey and Staehelin,
1986). I did not observe any recognizable membrane disruption or shrinkage due to

the dextran. Artifacts in samples of this study were observed as some ice crystal

55

damage which was easily identiﬁed and a separation ofthe lumen in the nuclear
envelope which was presumably the result of resin inﬁltration.

For many years it has been assumed by some investigators that protoplasmic
streaming in P. polycephalum occurs by a "gel-like" ectoplasm providing the motive
force for the "sol" or ﬂuid-like endoplasm. In this study I was interested in
determining whether the presence of an endoplasm and ectoplasm as described
previously was indeed real. The presence ofa gelliﬁed ectoplasm was described
previously (Wohlfarth-Bottermann, 1963,1964; Wolf et al., 1980; Kessler, 1982; and
Sauer, 1982) as being derived from numerous and extensive invaginations of the
plasma membrane around the veins. Previous studies documented the presence of
membranous blebs, whorls, and invaginations due to conventional chemical ﬁxation
techniques (Chandler, 1979; and Willison and Brown, 1979). Following
glutaraldehyde ﬁxation, membranes can continue to bleb and change (Aldrich,
1989). Rhea (1966) noted that the gelliﬁed ectoplasm was most likely an illusion of
undetected streaming in the peripheral region of the plasmodia. He noted that the
so called "gel" state appeared the same as the channel "sol" state ultrastructurally.
The results of this LTSEM study showed no indication ofa separate "gel-like"
ectoplasm. Glutaraldehyde ﬁxed plasmodia showed extensive damage and
invaginations which is presumed to be an artifact of ﬁxation.

For years it has been accepted that actin and myosin provide the motive
force in P. polycephalum for protoplasmic streaming, ever since Wohlfarth-

Bottermann (1962) ﬁrst described ﬁbrils in the plasmodium. These ﬁlaments, as

56

seen in this study, often were associated parallel to the plasma membrane on the
cytoplasmic side and adjacent to the plasmalemmal invaginations on the
cytoplasmic side. Other researchers which have demonstrated these ﬁlaments
ultrastructurally include: Wohlfarth-Bottermann (1962, 1963, and 1964), McManus
(1965), McManus and Roth (1965), Rhea (1966), Daniel and Jarlfors (1972a),
Haskins and Hinchee (1974), Charvat et al. (1973), and Wolf et al. (1981a,b).

Biochemical studies have been done which estimated the amount of actin and
myosin in the cytoplasm ofthe plasmodium compared to skeletal muscle (Kessler et
al., 1976). Kessler et. al. (1976) demonstrated that myosin accounts for 0.8% of the
total protein in the plasmodium as compared to 38% in skeletal muscle and actin is
estimated to comprise 15-25% ofthe total protein in the plasmodium, as opposed to
23% of muscle protein. These researchers concluded that the plasmodium has a
very high concentration of actin, with an actin to myosin ratio of 200:1 compared to
a 7:1 actin:myosin ratio in muscle.

Kessler (1982) discussed the possible involvement of a calcium-sensitive
actomyosin constriction system in the plasmodium. It is believed that calcium
concentration in the plasmodium is related to the mechanism for controlling
protoplasmic streaming in P. polycephalum. According to Kohoma et al. (1992),
calcium has been demonstrated to exert an inhibitory effect on actomyosin
polymerization. As free Ca3+ increases in the cytoplasm, actin disassembly is
activated through fragmin, an F-actin-fragmenting protein (Uyeda and Furuya,

1990; and Stossel, 1993), which corresponds to the contracted regions of the

57

plasmodium. Likewise, as free Ca2+ decreases in the cytoplasm, actin polymerizes,
corresponding to the relaxed regions of the plasmodium (Kessler, 1982).

It appears that the release and uptake of Ca2+ is an important part ofthe
regulatory mechanism for protoplasmic streaming in P. polycephalum (Kessler,
1982). Sauer (1982) discussed three mechanisms for reducing free Ca2+
concentration in the ground cytoplasm: pumping Ca2+ across the plasma
membrane, however, this is an inefﬁcient process considering that the external Ca1+
concentrations are 1000-fold higher than inside; ATP induced pumping Ca2+ into
vesicles; or and ATP-dependent reaction incorporating Ca1+ into mitochondria.
However, Sauer (1982) noted that the accumulation of Ca2+ in mitochondria has
been shown to be a slow, involved process.

In this HPF-FS TEM study, calcium presumably was observed as electron
dense deposits in vesicles and as mitochondrial inclusions. This supports the study
by Daniel and Jarlfors (l972b) in which calcium vesicles were observed by TEM and
identiﬁed using X-ray spectroscopic analysis and by Wolf and Stockem (1979).

The amoebal stage of P. polycephalum contains a microtubular, pro-flagellar
apparatus and microtubules for open mitosis with centrioles, however, the
plasmodial stage for years was believed to contain only microtubules in the mitotic
apparatus, which is semi-closed (Wright, 1982). It has been proposed many times
over the years that there is no extensive microtubular network due to the constant
movement and flow of the mature streaming plasmodium. Many investigators have

tried to demonstrate the existence of microtubules in P. polycephalum.

58

Microtubules were observed in very small plasmodia of Perichaena vermicularis
(Charvat et al., 1973), Ceratiomyxafruticulosa (Scheetz, 1972), and Physarum
polycephalum (Solnica-Krezel et al., 1990). McManus and Roth (1965) reported to
have seen microtubules in mature samples of Physarum melleum, however, their
ﬁxation protocol and interpretation of micrographs did not provide convincing
evidence.

Then in 1991, Salles-Passador et al. demonstrated microtubules in mature
plasmodia ofP. polyceplmlmn. However, they were only able to demonstrate
microtubules following an extensive chemical ﬁxation protocol containing PIPES,
HEPES, EGTA, magnesium, and Triton X-100 for TEM. Plasmodial samples for
their immunoflurescent study were ﬁxed for 15 minutes in cold methanol (-20°C)
which presumably could result in a high degree of ice crystal damage, creating a
high level of background ﬂuorescence. Salles-Passador et al. (1991) were not able to
collaborate their TEM and immunofluorescent studies, as their specimens ﬁxed for
TEM were not shown to contain microtubules by immunoﬂuorescence.

Samples prepared for this study by HPF-FS for TEM were shown to contain
cytoplasmic microtubules (M. L. C. Czymmek, H. C. Aldrich, and K. L.
Klomparens, manuscript in preparation), although they were sparse. They also
proposed that plasmodial microtubules may be more labile than other types which
easily may be preserved ultrastructurally (M. L. C. Czymmek, H. C. Aldrich, and K.

L. Klomparens, manuscript in preparation).

5 ‘)

The glycocalyx of P. polycephalum is primarily composed of an acidic
polysaccharide containing galactose with sulphate and rhamnose (McCormick et al.,
1970a; and Kessler, 1982). This slime layer, which also extends into the lumen of the
plasmalemmal invaginations, is believed to protect the plasmodium from external
ionic ﬂuctuations and may be involved in the enrichment of extracellular Ca7+
involved in protoplasmic streaming (Wolf et al. 1981a; and Kessler 1982). It has
been noted by Wolf et al. ( 1981a) that the golgi may serve a roll in the packaging of
mucous substances for the production ofthe slime layer.

Several researchers (Rhea, I966; Haskins and Hinchee, 1974; and Wolf et.
al., 1981a) have observed external microﬁlaments parallel to the plasma membrane
embedded in the glycocalx, however, this material, as noted by Wolf et al. (1981a),
has been difﬁcult to observe in the past by conventional chemical ﬁxation due to its
sensitivity to chemical ﬁxation and embedding procedures. The glycocalyx from the
HPF-FS samples for TEM in this study often contained ice crystal damage but these
small microﬁlaments as described previously were seen.

Haskins and Hinchee (1974) described ﬁlaments with conventional chemical
ﬁxation for both TEM and SEM. Their TEM studies seemed to correlate with those
studies of extracellular ﬁlaments as described above. However, their slime ﬁlaments
as described from SEM studies were much larger and were not seen running directly
parallel to the plasma membrane on the glycocalyx side. In this study, the larger
slime ﬁlaments as described by Haskins and Hinchee (1974) for SEM were observed

in both the HPF-FS samples for TEM and the LTSEM material. This is the ﬁrst

()0

time that these slime ﬁlaments have been demonstrated by TEM. These slime
ﬁlaments may increase the surface area of the plasmodium for increased absorption
or may serve as anchoring structures with the substrate.

Mitochondria prepared by HPF-FS for TEM in this study always were
observed as spherical, unlike the typically oblong mitochondria described in the
previous conventional chemical ﬁxation studies. In a chemical ﬁxation study by
Kuroiwa et al. (1994) it was proposed that mitochondria in P. polycephalum were
elongated throughout their life cycle, except for immediately following division.
When mitochondria were observed in the living state by phase contrast light
microscopy (M. L. C. Czymmek, H. C. Aldrich, and K. L. Klomparens, manuscript
in preparation) they had round proﬁles. P. polycephalum mitochondria may
become oblong following chemical ﬁxation and dehydration as the mitochondrial
matrix assumes the shape of the rod-shaped nucleoid (Aldrich, personal

communication).

CHAPTER II

LOW-TEMPERATURE SCANNING ELECTRON MICROSCOPY

O F PH YSA R Ugll POI. YCEPHA I. UM S PO RA N G IA

ABSTRACT

Low-temperature scanning electron microscopy allowed excellent
preservation of whole and cryo-fractu red sporangia of Physarumpolycephalum.
Frozen-hydrated sporangia examined with low-temperature scanning electron
microscopy showed the peridium of sporangia initials was very wrinkled, however,
at maturity it appeared much less convoluted. Small openings were observed on the
outer surface ofthe peridium in sporangial initials and mature sporangia.
Fractured samples revealed a double-walled peridium and many details within
sporangia including capillitial threads and lime nodes of various shapes and sizes.
Double-walled sporangiospores were ornamented with small spinules and many
appeared to contain a large depression. The spores were eventually released by

dehiscence of the calcareous peridium.

6|

()2

INTRODUCTION

The reproductive stage of Physarum polycephalum, as well as other members
of the Physarales, is characterized by the formation of sporophores from the
assimilative phaneroplasmodium under conditions of light and lowered nutrient
levels (Raub and Aldrich, 1982), which eventually give rise to haploid spores. The
sporophore type consists of sporangia which are formed according to subhypothallic
development (Alexopoulos, 1982). It is believed that the glycocalyx surrounding the
plasmodium froms a hypothallus in some species which serves as a base for the
sporangia. Sporangial initials are formed as small mounds on the plasmodial
surface which eventually elongate and constrict at the base due to the accumulation
of food vacuoles in the basal region and secretion of ﬁbrous components to form the
stalk with a mature sporangium located at the top (Alexopoulos and Mims, 1979;
and Raub and Aldrich, 1982).

Each sporangium is surrounded by a double or triple-layered peridium,
which may or may not be covered with lime (calcium carbonate). The capillitium
within the sporangium forms a network of elastic ﬁlaments surrounding the spores
which may or may not be devoid of lime and serves as a means of controlling the
dispersion of spores (Alexopoulos and Mims, 1979). The presence or absence and
type of capillitium as well as spore color and ornamentation have been used as a
basis for delineating species.

Few studies on spore wall composition have been done, however, it is believed

that melanin is responsible of the dark color characteristic of many species,

63

including P. polycephalum (McCormick et al., 1970b). According to Aldrich (1967),
there is a single mitotic division preceding spore cleavage within the sporangium of
P. polycephalum. Once cleavage occurs, meiosis takes place approximately 24hr
later within the spore initial. Once the spores are released from the sporangium,
germination occurs in the Physarales by either the split or pore method.

Aldrich (1982) noted that due to the high water content of many structures in
the Myxomycetes, that most ultrastructural studies have been done on spores,
peridium, or capillitium which may be air dried or ﬁxed by conventional chemical
ﬁxation techniques. Many artifacts have been associated with using conventional
chemical ﬁxation such as shrinkage of cells when using glutaraldehyde as well as
changes in cell volume following chemical dehydration along with other preparation
procedures of scanning electron microscopy (SEM) and transmission electron
microscopy (TEM) (Lee, 1984).

This study examined sporangial development in P. polycephalum using low-
temperature scanning electron microscopy (LTSEM) to determine the accuracy of
previous conventional chemical ﬁxation or air dried techniques. Due to excellent
preservation, additional information was obtained compared to earlier studies on
sporangia and other resistant structures in P. polycephalum (Aldrich, I967; Aldrich
and Blackwell, 1976; Alexopoulos, 1982; Chet and Kislev, 1973; Kislev and Chet,
1973; 1974; Randall and Lynch, 1974; Raub and Aldrich, 1982; Schoknecht, 1975;

and Schoknecht and Keller, 1989).

64

MATERIALS AND METHODS

Cultures ofP. polyceplmlum (Carolina Biological Supply) were grown on a
thin layer of 2% water agar under continuous light and lowered nutrient levels.
Small agar squares (4x4mm) with sporangia attached were excised and mounted on
a cryo—holder coated with a thin layer of Tissue Tek ll (Lab-Tek Products, Division
of Miles Laboratories, Naperville, II 60540) and graphite. The cryo-holder with
samples were rapidly plunged into a slurry ofliquid nitrogen and transferred onto
the JEOL 35CF scanning electron microscope cryo-stage and etched by slowly
warming to -65°C until all surface ice was removed (typically 5-10 minutes). The
samples on the cryo-holder were then cooled to -l30°C and transferred to the
EMscope SP2000 Sputter-cryo System where they were gold coated and then
transferred back to the JEOL 35CF for observation at -80°C and 10 kV. Fractured
samples were struck with a cooled cryo-knife after being plunged rapidly in liquid
nitrogen and then processed for low-temperature scanning electron microscopy as

described above.

()5

RESULTS

Frozen-hydrated sporangia at various stages of development were chosen and
examined by LTSEM which allowed observations of whole sporangial initials at
much earlier stages than previously recognized using conventional chemical ﬁxation
or air dried techniques. Wrinkled sporangial initials were observed as small papillae
without fully constricted stalks (Figs. 40 and 41). Close inspection revealed that
although the whole surface was convoluted, regions varied from smoother to highly
reticulate. Even though attempts were made, no correlation could be made
regarding the highly convoluted regions of the sporangial initial (greater surface
area) and the lobes of the mature sporangia.

Sporangial initials became much less convoluted as they matured (Fig. 42).
Figure 42 also illustrated multi-Iobed sporangia, developed by subhypothallic
growth, were supported on a single stalk made up of many folds on the hypothallus
base. The clustered sporangia appeared to contain many evenly distributed small
openings on the calcareous peridial surface (Figs. 42-44) and numerous spore initial
outlines.

Irregular dehiscence of the peridium occurred as the sporangia contained
cracks that were not a result of preparation procedures for LTSEM (Figs. 45-48)
and were consistent with previously reported conventionally ﬁxed samples. Cryo-
fractured LTSEM of sporangia initials revealed a nucleus and other cytoplasmic
material (Fig. 49) and a double-walled peridium with a thick, calcareous outer

membrane and a more delicate inner membrane (Fig. 50). Although the spore

()6

initials fractured smoothly the sporangiospore protoplasm exhibited evidence of
freeze damage indicating a higher water content.

Sporangiospores were ornamented with many small spinules and many
contained a large depression which was previously described in other conventional
chemical ﬁxation studies as a dehydration artifact (Figs. 51 and 52). However, it is
now believed that the large depression is a result of normal desiccation conditions
and the naturally dried collapsed spore appearance was not a result of LTSEM. A
cryo-fractured LTSEM sample revealed double-walled sporangiospores (Fig. 53).

The capillitium appeared as a network of tubes connecting fusoid and
elongated lime nodes with sporangiospores inside sporangia (Figs. 51, 52 and 54).
The tube-like and sheet-like portions ofthe capillitium expanded following

dehiscence of the peridial surface and sporangiospores were released (Fig. 54).

 

67

FIG. 40. LTSEM of Physarum polyceplmlum sporangial initials. Excellent
preservation of2 sporangial initials with LTSEM showed wrinkled peridial surfaces

(arrows). Bar = 100 um.

Ir.-f.”1 -r~~-;4"..y-"'..-: ,: L.

so. ' '

‘1 ‘ . . 4‘ 4.
__. _ ’ w ‘4‘-
“ 7 ~71“: 3.7".)
_. 29".. “I’D... '
o.‘--’ an" '
-.r g-‘
- .‘f-

..
..
," f

 

() ‘)

FIGS. 41-42. LTSEM of Physarumpolycephalum sporangia. Fig. 41. High
magniﬁcation of Fig. 40 showed a well preserved, wrinkled peridium (arrows) of a
sporangial initial. Bar = 20 um. Fig. 42. The surface of more mature sporangia
appeared much less convoluted than that of Figs. 40 & 41 and contained many small
openings (arrowheads). Note hypothallus (H) and stalk (S) which has fully

constricted. Bar = 500 pm.

 

70

 

 

71

FIGS. 43-44. LTSEM of Physarum [mlycephalum sporangia. Fig. 43. A
more mature sporangium with many small openings (arrowheads) and cracks
(arrows) in the peridium. Bar = 100 um. Fig. 44. A higher magniﬁcation of Fig. 43
showed the irregular shaped openings (arrowheads) in the peridium. The outline of

the spores (S) were observed beneath the peridium. Bar = 10 um.

72

 

 

 

73

FIG. 45. LTSEM of Physarum polycephalum mature sporangia. The
sporangia at this stage appeared to contain cracks (arrows) in the surface of the

peridium. Bar = 250 um.

 

75

FIGS. 46-48. LTSEM of Physarum polycephalum peridial surface. Fig. 46
shows a much later stage in sporangial development showed extensive cracks in the
peridium (arrows) with many spores (S) under the surface. Bar = 200 um. Fig. 47
represents an earlier stage in sporangial development compared to Fig. 48. Note the
openings (arrowheads), spores (S), and cracked peridium (arrow). Bar. = 20 um.
Fig. 48 shows a later stage of development in which there were a larger number of
cracks in the peridium. Note the opening (arrowhead) and the spores (S). Some
spores at this stage contained a large depression (D) which presumably was the
result of natural conditions (desiccation), not preparation (dehydration shrinkage).

Bar = 20 um.

   

77

FIGS. 49-50. Cryo-fractured LTSEM of Physarum polycephalum sporangia.
Fig. 49. High magniﬁcation of cryo-fracture sporangia revealed spore initials (S)
surrounded by protoplasm which was excluded from the spore initials. Bar = 10
um. Fig. 50. Cryo-fractured sporangia with a doubled-layered peridium
(arrowheads) revealed many details of development. Note the spore initials (S),

protoplasm (arrows), and nucleus (N). Bar = 20 um.

78

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FIGS. 51-52. LTSEM of Physarumpolycephalum spores. Fig. 51. High
magniﬁcation of a later stage of spore development. The spores (S) were
ornamented with small spinules (arrow) and were closely associated with the
capillitium (C). Bar = 5 pm. Fig. 52. Many spores (S) at this stage of development
contained a large depression (D) which was not the result of dehydration artifact

due to preparation. Note the capillitium (C). Bar = 10 pm.

80

 

8|

FIGS. 53-54. LTSEM of Physarum polycephalum spores and capillitia. Fig.
53. Cryo-fractured, more mature sporangia (than Fig. 49 - 50) showed the two wall
layers (arrows) surrounding the spore protoplasm (P). Note the irregularly shaped
spores (arrowheads). Bar = 10 um. Fig. 54. Very mature sporangia in which the
peridium appeared to deteriorate (not cryo-fractured). Note the naturally

desiccated spores (s) and capillitium (c). Bar = 50 um.

82

 

83

DISCUSSION

LTSEM proved very effective in preserving surface morphology and
maintaining spatial relationships in sporangia ofP. polycephalum. LTSEM rapidly
immobilizes dynamic biological processes with no exposure to chemicals which may
leach out components and swell or shrink the cells (Beckett and Read, 1986).
Sporangial initials, due to their higher water content than mature sporangia, were
often difﬁcult to preserve using conventional chemical ﬁxation or air dried
techniques. LTSEM allowed observations of Sporulation at earlier stages for P.
polycephalum than obtained previously for SEM (Alexopoulos, 1982; Kislev and
Chet, 1973; Randall and Lynch, 1974; Schoknecht, 1975; and Schoknecht and
Keller, 1989).

Sporulation in P. polycephalum resulted in the formation ofa sporangium
with a double-layered peridium. The outer peridial covering is characterized by
amorphous, granular deposits of calcium carbonate with some calcium phosphate,
as determined previously using energy dispersive microanalysis (Schoknecht, 1975;
and Schoknecht and Keller, 1989).

The extent to which the calcium carbonate deposits are formed on the
peridium is often determined by environmental conditions (Alexopoulos, 1982; and
Schoknecht and Keller, 1989). Sporangial samples in this study did not appear to
contain many large amporphous granular calcium carbonate deposits on the
peridium compared to those seen by Schoknecht and Keller (1989) and may be

attributed to the lower calcium concentration in the agar. In this LTSEM study the

 

X-l

peridial surface could be seen closely appressed with the outline of the spores and
the view was not obstructed by the calcium deposits.

Sporangial initials as well as mature sporangia contained many evenly
distributed holes on the outer calcareous peridial surface. lt has been proposed
by Keller and Schoknecht (1989) and Schoknecht and Keller (1989) that these holes
in the peridium serve as "nucleation sites" for calcium carbonate deposition on the
peridial surface and that these "peridial pores" may determine where the calcium
carbonate is eventually deposited. Kislev and Chet (1973) noted that the capillitium
is comprised of interconnecting tube-like structures connected to the holes in the
peridium through which calcium carbonate may be excreted to the outer surface of
the sporangium. Due to the low presence of amorphous calcium carbonate deposits
on the sporangial surface in this study these observations could neither be conﬁrmed
nor denied.

Calcium is concentrated in the mitochondria, vesicles, and membrane
surfaces (Schoknecht and Keller, 1989) ofP. polycephalum as discussed in chapter
one. There are a few theories on how the calcium then becomes concentrated in the
outer peridial layer. It is believed that the methods for deposition of calcium
carbonate differs for the Physaraceae and the Didymiaceae (Schoknecht and Keller,
1989). Gustafson and Thurston (1974) and Collins (1979) both support the notion
that in the Didymiaceae, the calcium is secreted on the peridial surface directly in
ionic form through the plasma membrane (Schoknecht and Keller, 1989). Bechtel

and Homer (1975) preposed that calcium was deposited on the peridium by way of

 

85

vacuoles that fused with the plasma membrane. It was also noted that the "calcium
granules" were ﬁrst excreted from the vacuoles internally as "minute spheres" and
then deposited on the peridial surface (Bechtel and Homer, 1975; Collins, 1979; and
Schoknecht and Keller, 1989).

Schoknecht and Keller (1989) postulated that the way in which the calcium is
excreted will eventually affect the shape of the calcium carbonate and calcium
phosphate deposits. This is very possible in light of the fact that the majority of
calcium carbonate deposits seen in the Didymiaceae are stellate crystalline whereas
those in the Physaraceae are most often amorphous and granular (Shoknecht, 1975;
and Schoknecht and Keller, I989). Unlike the Didymiaceae, members of the
Physaraceae contain phosphorous in the from of calcium phosphate in their peridial
deposits which also may affect the crystalline structures leading to an amorphous
appearance due to the calcium carbonate dissolving in the alkaline environment
(Schoknecht, 1975; and Schoknecht and Keller, 1989).

LTSEM was useful in observing the individual spore wall layers in P.
polycephalum which represented the ﬁrst study of SEM in which they could be seen.
The double-walled spore contained an electron transparent, ﬁbrous layer and an
electron opaque, granular layer which was earlier observed by TEM (Randall and
Lynch, 1974). Spinules were formed early on in wall development (Randall and
Lynch, 1974). According to Aldrich (1967) a single mitotic division occurs
preceding spore cleavage with meiosis taking place approximately 24h later inside

the spore initial.

86

Composition ofthe spore walls in P. polycephalum was studied by
McCormick et al. (1970b) and were shown to contain galactosamine, 2% protein,
15% melanin, and some phosphate. It was noted that sporulation involved new
synthesis of RNA, proteins, and polysaccharides (McCormick et al., 1970b) with
glycogen as the primary storage reserve within the spores (Aldrich and Blackwell,
1976)

Previous reports suggested that the collapsed appearance of the mature
spores was an artifact probably due to improper dehydration (Kislev and Chet,
1973), however, these spores were also observed in this LTSEM and presumably due
to natural desiccation conditions supporting the ﬁndings of Schoknecht and Small
(1972). Dehydration artifacts are not artifacts associated with LTSEM (Beckett and
Read, 1986).

Low temperature scanning electron microscopy of sporangiophore
development in P. polycephalum has provided additional insights regarding the
nature of sporangiophore maturation. LTSEM was successfully used during the
plasmodial stages as well. This technique, when employed with care, can yield

signiﬁcant improvements in preservation of slime molds as described in this study.

SUMMARY

This study examined the ultrastructure of Physarumpolycephalum using
high-pressure freezing and freeze-substitution in conjunction with transmission
electron microscopy and plunge freezing with low-temperature scanning electron
microscopy. These techniques proved very elTective in obtaining morphological and
ultrastructural details of plasmodial and sporangial devel0pment. Unlike the
conventional chemical ﬁxation studies described previously, this study enabled
cellular components to be rapidly immobilized, thus eliminating pre-exposure to
chemical ﬁxatives which often cause signiﬁcant modiﬁcations prior to cellular
cessation.

The plasmodium has been difﬁcult to preserve ultrastructurally due to its
very high water content which makes it susceptible to ﬁxation and dehydration
artifacts as well as mechanical damage from specimen preparation procedures.
Mechanical damage due to sample manipulation prior to freezing was minimized for
high-pressure freezing and freeze-substitution with transmission electron
microscopy by growing the plasmodia in the high-pressure freezer gold hats prior to
freezing. This method of preparation resulted in excellent preservation of
ultrastructural details with minimal ice crystal damage. Plasmodia examined by

transmission electron microscopy revealed smooth membrane surfaces throughout

87

 

88

the cytoplasm. Spherical mitochondria were observed, unlike those seen in previous
conventional chemical ﬁxation studies. Many vacuoles of various shapes and sizes
were observed along with contractile vacuoles, food vacuoles, nuclei, golgi, and
round endoplasmic reticulm vesicles.

Plasmodial details obtained by low-temperature scanning electron
microscopy provided information on general surface topography including the
glycocalyx, plasmodial veins and slime ﬁlaments. Plasmodial strands appeared to
emanate from different levels to form the plasmodial body.

Sporangia examined by plunge freezing with low-temperature scanning
electron microscopy revealed morphological details such as a wrinkled peridium of
sporangial initials which later appeared as smooth contoured peridium in mature
sporangia. Small openings were observed on the outer calcareous peridial surface of
all sporangia observed. The capillitial network surrounded the sporangiospores and
appeared as tubes with fusoid and elongated lime nodes. Sporangiospores were
double-walled and many appeared to contain a large depression. Dehiscence of the
sporangial wall occured as irregular cracks in the peridial walls.

Cryo-techniques have provided an effective method of cellular preservation
in Physarum polycephalum. These techniques are probably broadly applicable to
many other Myxomycetes as well as other organisms with plasmodial stages. With
these improved methods of cellular preservation, more reliable interpretations of

ultrastructural data may be obtained.

 

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