J. Cell Sci. 10, 563-583 (1972)
Printed in Great Britain
563
MOVEMENT, FINE STRUCTURE, AND FUSION
OF PSEUDOPODS OF AN ENCLOSED AMOEBA,
DIFFLUGIELLA SP.
J. L. GRIFFIN
Department of Anatomy, Harvard Medical School, Boston, Massachusetts 02115, and
the * Armed Forces Institute of Pathology, Washington, D.C. 20305, U.S.A.
SUMMARY
In Difflugiella sp., strain F-20, a small amoeba enclosed in a flexible mantle, pseudopods
extended through a mouth or aperture and seemed to function only for movement and feeding.
Pseudopods from different cells fused on contact and cell clumps shared common pseudopods
and moved in a co-ordinated way. During locomotion, pseudopods or pseudopod complexes
usually exhibited an activity cycle of 3 phases: anterior extension with the tip firmly adhering;
stable hold as other pseudopods advanced; and flaccid posterior retraction. While distal
adhesive tips advanced, proximal unattached parts of pseudopods simultaneously shortened as
the cell body advanced.
Microtubules were numerous in pseudopods within the mouth but extended for only
1-2 /an into pseudopods up to 20-30 /an long. Microfilaments were present where pseudopods
adhered to the substratum, to the mantle, or to bacteria and were also associated with
pinocytotic invaginations. Pseudopod ground plasm was either reticulate or amorphous; no
axial rods or aligned filaments related to pseudopod rigidity were seen.
Simultaneous pseudopod adhesion, extension, and proximal shortening apparently account
for locomotion or cell body translation of Difflugiella. While some similarities to other amoeboid
systems were noted, the need for detailed studies on different types of organisms or cells is
emphasized.
INTRODUCTION
Much recent evidence indicates that actin is present in Acanthamoeba (Pollard,
Shelton, Weihing & Korn, 1970), Amoeba (Pollard & Korn, 1971), acellular slime
moulds (Hatano, Totsuka & Oosawa, 1967; Adelman & Taylor, 1969; Nachmias,
Huxley & Kessler, 1970), cellular slime moulds (Woolley, 1970), and metazoan cells
(Ishikawa, Bischoff& Holtzer, 1969). Myosin is also present in slime moulds (Hatano
& Ohnuma, 1970; Adelman & Taylor, 1969; Nachmias & Ingram, 1970) and may be
in thick filaments in amoebae (Pollard & Ito, 1970; Holberton & Preston, 1970).
Although amoeboid movement apparently involves energy-linked alterations of
organized substructure, the new molecular information does not necessarily clarify
the relationship between motility and visualized fine structure.
Bundles of actin microfilaments in the slime mould Physarum polycephalum are
appropriately located to contract and squeeze out streaming cytoplasm (WohlfarthBottermann, 1964a, b; Rhea, 1966; Porter, Kawakami & Ledbetter, 1965; Nagai &
• Present address.
564
J. L. Griffin
Kamiya, 1966), but the presence of filament bundles large enough to be seen by
polarizing microscopy is inversely related to streaming and vigorously streaming
plasmodia may have none (Nakajima, 1964; Nakajima & Allen, 1965); aggregates
appear during pinocytosis (Bauer, 1967; Griffin, 1967).
Filaments of 2 sizes are present in amoebae fixed in locomotion (Pollard & Ito,
1970) but are more numerous in non-motile, pinocytosing amoebae [Chaos and
Amoeba) and in amoebae broken during fixation (Nachmias, 1964; Griffin, 1965).
Numerous filaments seen after enzyme treatments (Komnick & Wohlfarth-Botterman,
1965) are probably related to pinocytosis induced by the enzymes rather than to
better fixation permitted by membrane changes. Schafer-Danneel (1967) best
visualized filaments in amoebae fixed while cold and unable to move. In the above
systems, filament aggregates seem to differentiate under conditions in which movement slows and stops. Combining isolated thin (F-actin) and thick filaments from
A. proteus under appropriate conditions does produce dramatic movements (Wolpert,
1965; Pollard & Ito, 1970).
Filaments (about 6-nm) have been seen in the enclosed amoebae Difflugia
(Wohlman & Allen, 1968) and Hyalosphenia (Joyon & Charret, 1962) and a flabellulid
amoeba (probably Vannella) called Hyalodiscus simplex (Wohlfarth-Botterman,
1964a, b). Bhowmick (1967) visualized plasma filaments of about 20 nm in Saccamoeba
sp., strain F-13, Griffin; this amoeba also contains numerous 6-nm filaments (personal
observation). Various slime moulds (McManus & Roth; 1965), the giant amoeba
Pelomyxa palustris (Griffin, 1965; Daniels & Breyer, 1967), and other amoebae, naked
and shelled (personal observation), also contain microfilaments and microtubules,
which could play structural, contractile or other roles.
Microtubules seem to serve a structural or skeletal role in Heliozoa (Tilney &
Byers, 1969; Roth & Shigenaka, 1970), Foraminifera (McGee-Russell & Allen, 1971),
pigment cells (Bikle, Tilney & Porter, 1966) and blood cells (Fawcett & Witebsky,
1964), and microfilaments (F-actin) may have a similar role in microspikes and
microvilli (Taylor, 1966; Ishikawa et al. 1969) and acanthopodia (Pollard et al. 1970).
In cells of the plant Nitella, filament bundles lie parallel to the stream at the shear
interface (Nagai & Rebhun, 1966; Rebhun, 1967).
At present, correlated light- and electron-microscopic studies suggest direct
involvement of filament aggregates in one tension-generating system (Wohlman &
Allen, 1968) and an active shearing system (Nagai & Rebhun, 1966). Cytochalasin B
has recently been used to probe structure-function relationships. Wessells et al.
(1971) review the literature and unpublished work and conclude that this compound
reversibly inhibits contractile microfilaments in many systems and that reticulate
networks, not aligned aggregates, are contractile in individual cells. However,
membrane changes in pinocytosis influence microfilaments and cytochalasin can alter
cell-environment interactions (Carter, 19676), which suggests that secondary as well
as primary changes might be detected after application of this chemical. Studies of
cytochalasin effects at the molecular level should be interesting.
Comparative studies are needed because of the great diversity of parameters of
amoeboid movement (see Allen & Kamiya, 1964). It seems important to relate
Movement and fine structure of an enclosed amoeba
565
visualized fine structure to patterns of movement in living cells. Difflugiella sp. also
offers some advantages beyond that of a comparative approach. Organelles and
inclusions are enclosed within a capsule, from which pseudopods extend, so pseudopod
ultrastructure should reflect the functions of movement and feeding. Fixation of thin
pseudopods should be rapid, thereby improving the chances of preserving ultrastructure in a lifelike state.
MATERIALS AND METHODS
Difflugiella sp., strain F-20, Griffin, was isolated from a mixed culture of Amoeba proteus and
grown on i % non-nutrient agar with Aerobacter, strain A-154, added as food. An unidentified
spore-farming bacterium was present in these cultures. Assignment of F-20 to the genus
Difflugiella is based primarily on a discussion by Page (1966). A flexible test or mantle is
characteristic of this genus. Since F-20 is small and the pseudopods seem unique, further
studies may well justify a generic reassignment. Euglypha sp. used for comparisons is strain
F-54, Griffin.
For light microscopy, amoebae were usually observed in the inorganic medium of Prescott
& Carrier (1964) with Zeiss (Jena) phase and Nomarski optics. Movement was recorded by
cinematography or by sequential photographs, usually at 5-s intervals. An electronic flash
(Fawcett & Ito, 1958) was used for some still photography.
Amoebae were fixed at room temperature by rapidly pouring fixative over amoebae growing
and migrating on agar. The micrographs presented here were from amoebae fixed either with
2 % unbuffered osmium tetroxide plus 0002 M CaCl, for 5 min or with 125 % glutaraldehyde,
collidine 01 M, pH 77, with 0002 M CaCla for 2 h, followed by unbuffered OsO4 for 5 min.
The fixative is indicated in the caption for each figure. Some material was perhaps extracted by
the procedure used, but the calcium seemed to stabili2e components of interest (see Wood &
Luft, 1965, and Griffin, 1963). For in-block staining with uranyl acetate, cells were fixed for
1 h in 4% glutaraldehyde in 0-05 M PO4) pH 69, 20 min in OsO4 in 005 M PO4, rinsed twice
in 01 N sodium acetate and stained 20 min in 3 % aqueous uranyl acetate (Terzakis, 1968).
After fixation, agar wedges with adhering amoebae were cut out, dehydrated in ethanol, passed
through propylene oxide into Epon 812, and embedded open face. Sections were usually
stained with a saturated solution of uranyl acetate in water followed by lead citrate (Venablc &
Coggeshall, 1965).
RESULTS AND OBSERVATIONS
As in other enclosed amoebae, Difflugiella has a relatively stable organelle distribution. At the oral end, pseudopods extend from the specialized mouth (Figs. 1, 5-9)
near which contractile vacuoles are located (Figs. 1, 5). Granules and food or digestion
vacuoles form a band across the middle of the cell (Fig. 1). The nucleus, with a single
complex nucleolus, is located at the adoral end (Fig. 5). Clumps of fused cells (as in
Figs. 4 and 5) are sometimes made up of a dozen or more cells which share common
pseudopods and move in a co-ordinated way. No indications were seen of nuclear or
organelle exchanges between cell bodies.
Light-microscopic observations on movement
A detailed presentation of specific movement patterns is made in Figs. 1-4 and
their captions.
Commonly, several pseudopods or pseudopod complexes extended from beneath
an erect body, mostly extending within a half circle in the direction of locomotion,
remaining until newer, more anterior complexes had formed, then withdrawing from
566
J. L. Griffin
the region behind the cell body. Amoebae in locomotion showed characteristic
pseudopodial patterns (Figs. 1-4). On glass, pseudopods were frequently straight with
smooth outlines (Figs. 2-4), while more irregular outlines seemed to result from a
closer adhesion to agar. Thin webs frequently formed and retracted between pseudopods, particularly larger fused pseudopods (Fig. 4). In motion pictures, sequential
photographs and direct observations, only pseudopods extending in contact with the
substratum were seen to influence locomotion. F-20 can form free, unattached
pseudopods, but, as in flabellulid amoebae (Bovee, 1964), these are not involved in
locomotion. An extending pseudopod (moderate adhesion) was preceded by an
adhesive tip that was flattened, somewhat irregular, and relatively narrow (Figs. 1-4).
The cell body moved in a direction determined by the advance of multiple pseudopods
and the distance between the cell body and stationary branches of individual pseudopods shortened up to 50% while the tip was still extending (Figs. 1, 2). This early
proximal shortening was distinct in time and position from posterior withdrawal. As
a pseudopod became fully formed and reached a temporarily stable stage, it seemed to
thicken and straighten (Fig. 2, top). Withdrawing pseudopods were usually detached
andflaccid(Fig. 2B, c). Zipper-like fusion of adjacent pseudopods frequently preceded
retraction (Fig. 2C, D). Retraction was normally faster than extension (Fig. 4A, B);
pseudopods up to 30 fim in length sometimes withdrew within 1-2 s. The ingestion of
a bacterium seemed to involve a simple movement of adhering membrane over the
particle, without formation of a food cup (Fig. 4).
When pseudopods touched, fusion was immediate, whether they originated from
the same cell mass or from difFerent cells (Fig. 1). Although fusion occurred when
amoebae met, the possibility exists that aggregates may have derived in part from
divisions in which the cell body, but not the pseudopods, separated. The separation of
fused pseudopods seemed similar to normal retraction. Usually, as amoebae moved
apart, the fused pseudopod detached from the substratum. Such fused pseudopods
sometimes seemed to be under tension, but then parted slowly near the middle
without elastic rebound. Some contacts between pseudopods seemed to initiate an
immediate response that caused separation within 10—15 s after fusion (Fig. 1). Noncompressed amoebae did not differ in fusion and separation patterns from the gently
compressed cells in Fig. 1.
Ultrastructure of pseudopods
Electron micrographs showed that full cytoplasmic continuity was established
between cells sharing pseudopods (Fig. 5). Pseudopods contained few organelles or
granules, although mitochondria were seen near the mouth (Figs. 9, 14). Microtubules,
microfilaments, areas with a reticulate texture, and gradients in electron density were
seen within the pseudopods after fixation with osmium or glutaraldehyde (Figs. 6-9,
14-17). Although numerous (over 40 in one cross-section), microtubules were present
in pseudopods emerging from the mouth (Figs. 6—8), at a distance of 1 /*ra they were
indistinct (Figs. 6, 7, 14), and none were observed more than 2 /tm away from the
mouth (Figs. 6-9, 14-17). Some microtubules seemed to anchor the junction of the
membrane and the oral collar (Fig. 9).
Movement and fine structure of an enclosed amoeba
567
Microfilaments in pseudopods were usually near regions, presumably adhesive,
adjacent to the agar substratum (Figs. 9, 16), to the mantle of the amoeba (Figs. 8, 9)
or to bacteria (Fig. 15). Microfilaments also oriented towards membrane imaginations
(Fig. 9), but otherwise seemed to be present only at or near sites of adhesion.
The mantle surrounding the cell body is composed of 3 layers: (1) an external
electron-dense layer about 9-10 nm thick, (2) a directly underlying amorphous layer
of about 20 nm, and (3) just within the amorphous layer, punctate and circular profiles, possibly representing filaments and tubules in cross-section (Figs. 8, 9). At the
mouth, the mantle is continuous with the slightly more complex oral collar (Figs. 5—9).
The membrane at oral collar-membrane junctions stained intensely (Figs. 5-9) and
served as an attachment for 20-nm microtubules (Fig. 9).
In micrographs of most species of amoebae processed and photographed as in
Figs. 6-9 and 14-17, internal membranes usually appear as single lines, while thicker
external membranes are relatively easy to resolve as trilaminar 'unit membranes', as
shown in micrographs of Euglypha (Figs. 11, 12). In micrographs of F-20 a trilaminar
structure of external membranes is hard to detect (Fig. 13) except after uranium in
block (Fig. 10).
In other samples there is slight variation in dimensions as reported for Figs. 10—13,
but differences in both thickness and contrast are reproducible. The external membrane
on pseudopods and inside the mantle thus differs from that of most other small
amoebae in both structure and behaviour (fusion).
DISCUSSION
Patterns of movement
While it is clear that specimens of Difflugiella exhibit a distinct type or pattern of
amoeboid movement and characteristic fine structure, direct evidence related to
molecular interactions or mechanisms of movement is lacking. No internal or external
particles that might reveal relative movements of pseudopod protoplasm or membrane
were seen in Difflugiella, except during retraction. The dissociation of cellular systems,
useful in studies of other amoebae (Pollard et al. 1970; Pollard & Ito, 1970; Pollard &
Korn, 1971; Wolpert, 1965; Allen, Cooledge & Hall, i960; Griffin, 1964), was not
attempted.
The locomotion of Difflugia, a much larger enclosed amoeba, involves sequential
extension of unattached pseudopods, adhesion at the tip, and shortening to exert
traction (Wohlman & Allen, 1968; Mast, 1931). Because Difflugia drags a heavy shell
made of sand grains, its locomotory organelles or mechanisms are presumed to be
specialized for heavy work loads.
In Difflugiella, adhesion, pseudopod extension, and shortening (to exert traction ?)
occur simultaneously rather than sequentially. While the adherent tips of pseudopods
are still extending, proximal shortening decreases the distance between the cell body
and the stationary regions of attachment. Posterior withdrawal of flaccid pseudopods
occurs later and does not contribute to cell body advance. Proximal shortening during
pseudopod extension presumably accounts for cell-body translation in the direction
of locomotion.
568
J. L. Griffin
Because similarities are lacking, it seems unlikely that Difflugiella could utilize,
without modification, mechanisms proposed to account for movement of lobose
amoebae (Allen, 1961; Allen, Francis & Nakajima, 1965; Alien & Kamiya, 1964;
Jahn & Bovee, 1969; Noland, 1957) or Foraminifera (McGee-Russell & Allen, 1971;
Allen, 1964; Jahn & Rinaldi, 1959).
Difflugiella can form relatively rigid free or unattached hyaline pseudopods. These
may be analogous to the hyaline pseudopods of Vannella and other mayorellids (Bovee,
1964), although their movements are not as complex. The free pseudopods of
Difflugiella do not contribute to locomotion. In locomotion, all extending pseudopods
adhere and normally exhibit a finely irregular or fuzzy appearance at the advancing
tipA significant feature of movement of Difflugiella is the close relationship between
pseudopod extension and adhesion, a correlation also seen in Vannella and other
amoebae normally led by a flattened anterior fan (Bovee, 1964). In these amoebae,
the membrane moves forward over the cell and remains stationary under the advancing
body like a tank tread (Griffin & Allen, i960; Griffin, 1970). In an abstract (Griffin,
1970), a modified frontal contraction (see Allen, 1961; Allen et al. 1965; Allen &
Kamiya, 1964) was suggested as compatible with the advance of the adhering hyaline
fan of Vanella. This conceptual model also seems compatible with the advance of the
adhering tip of hyaline Difflugiella pseudopods, but it is quite possible that the
compatibility is apparent only because little direct evidence is available. Of course,
internal consistency and compatibility with available evidence are not enough to
prove a theory correct. Metazoan cells also show a close correlation between adhesion
and extension of hyaline peripheral fans (Taylor, 1966; Buckley & Porter, 1967;
Carter, 1967a; Wessells et al. 1971).
Possible functional roles of visualized fine structure
It seems reasonable to assume that the fine structure visualized was present in the
living state. Light microscopy revealed no gross distortion and the amoebae were
moving normally just prior to rapid fixation at room temperature. The thin pseudopods
and simple membranes of Difflugiella should present no special problems in fixation.
It is encouraging that fixation with either glutaraldehyde or osmium preserved
essentially similar patterns in pseudopods of Difflugiella. A second assumption is that
the activity of the cell at the instant of fixation was correctly inferred from the
morphology of the embedded cell, and from light- and electron-microscopic sections.
The cells sectioned were not directly visualized during fixation and processing
(Griffin, 1963).
Ground cytoplasm of pseudopods. Regions of pseudopods interpreted as ground
cytoplasm (not adjacent to adhesive regions) appeared either amorphous or reticulate,
with reticulate filamentous elements less than 5 nm in diameter. The difference between reticulate and amorphous areas may reflect either different physical states of the
protoplasm or merely differences in preservation and visualization. For example,
complete preservation and staining of soluble proteins could make it impossible to
visualize a diffuse reticulate ground structure.
Movement and fine structure of an enclosed amoeba
569
The resistance to deformation of pseudopods seems to be based on a reticulate
substructure, rather than on components such as microfilaments or microtubules. The
substructure of Difflugiella pseudopods seems not to differ in any significant way from
the protoplasmic substructure of hyaline regions of many different cell types.
Microfilaments. Relatively straight microfilaments of about 6 nm were seen near
where membranes adhered to the substratum, the mantle, or bacteria and in regions
of pinocytosis. They seemed not to be involved in maintaining configuration of
elongate pseudopods or other structures. In many micrographs a gradual transition
of cytoplasmic texture was seen, ranging from a diffuse reticulate background to more
distinct microfilaments adjacent to apparent areas of adhesion. Microfilaments may
be an alternative configuration of reticulate material.
The microfilaments seem analagous to those formed in larger amoebae in response
to pinocytosis inducers, such as alcian blue (Nachmias, 1964) and enzymes (Komnick
& Wohlfarth-Botterman, 1965), and may reflect a cytoplasm-membrane bonding
required either for pinocytosis or to reinforce a site of adhesion to the substratum.
Taylor (1966) saw both microfilaments and microtubules adjacent to sites of adhesion
of tissue culture cells. Light-microscopic views of pseudopod complexes of Difflugiella
(as in Fig. 4) with webs between the pseudopods look like webs between stress fibres
in tissue culture cells (Buckley & Porter, 1967), but fine-structural similarities were
not seen. Thick microfilaments (Pollard & Ito, 1970) were not seen.
Microtubules. Microtubules in the mouth of Difflugiella could act as skeletal elements
or support translational machinery for moving vacuoles through the mouth (compare
Rebhun, 1967). Microtubules anchoring the collar-membrane junction probably help
to maintain the configuration of the cell body. Microtubules were not seen in pseudopods so apparently do not contribute to pseudopod rigidity, as in heliozoans (Tilney &
Byers, 1969; Roth & Shigenaka, 1970) and foraminiferans (McGee-Russell & Allen,
1971). The microtubules are clear and distinct only in the region within 1 fim of the
mouth, suggesting that something in that region may act as an organizer for microtubule differentiation. Their absence in pseudopods does not seem to be a fault in
preservation, since both types of fixative give the same distribution.
Membrane. The membrane of Difflugiella differs from external membranes of most
amoebae both in behaviour and in thickness and contrast. Immediate fusion was seen
whenever pseudopod contact was observed. In most species of amoebae, the external
membrane shows clear differentiation into a trilaminar' unit membrane' configuration.
A trilaminar configuration was seen in the external membranes of Difflugiella only
after uranyl acetate in block or when osmium-fixed material was sectioned very
carefully.
Cellular fusion is important in many examples of morphogenesis, but reversible
fusion of pseudopods only has apparently not been reported. The larger pseudopods
of clumps may permit more effective food gathering or locomotion under certain
circumstances.
570
J. L. Griffin
CONCLUSIONS
That pseudopod adhesion, extension, and shortening account for locomotion is not
surprising, since it is difficult to imagine any amoeboid progression without similar
processes. Although micronlaments were adjacent to sites of membrane adhesion,
neither micronlaments nor microtubules seem involved in other pseudopodial
function. Reticulate pseudopod substructure may represent some aspect of the
machinery for movement, if such machinery was preserved and visualized. This
conclusion would be compatible with concepts advanced by Wessells et al. (1971).
Note. Some of the material herein was presented to the American Society of Cell Biology
(Griffin, 1968). In a recent review, Jahn & Bovee (1969) wrote 'This stereoplasmic track in
Difflugiella is a bundle of micronlaments that develop in only the attached portions of the
filopods (194).' Although reference 194 is to Griffin (1968), there is no stereoplasmic track in
Difflugiella and no known reason for their statement.
This study was supported in part by NIH Research Grant AIO 3410, an NIH Special
Fellowship, an NIH Departmental Training grant, and small grants from the Milton Fund
(all at Harvard Medical School); and a Research Contract, Project No. 3A062110A822, from
the Medical Research and Development Command, U.S. Army, Washington, D.C. The
opinions or assertions contained herein are the private views of the author and are not to be
construed as official or as reflecting the views of the Department of the Army or the Department
of Defense.
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MAST, S. O. (1931). Movement and response in Difflugia with special reference to the nature
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NACHMIAS, V. T., HUXLEY, H. E. & KESSLER, D. (1970). Electron microscope observations on
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NAGAI, R. & KAMIYA, N . (1966). Movement of the myxomycete plasmodium. I I . Electron
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Ultrastruct. Res. 14, 571-589.
NAKAJIMA, H. (1964). T h e mechanochemical system behind streaming in Physarum. In
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[Received 21 June 1971)
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Fig. i A, B. Two sequential phase-contrast photographs of living Diffktgiella sp. gently
compressed against soft agar so that locomotion continued. In each cell, pseudopods
emerged from the mouth and extended in close contact with the agar. Contractile
vacuoles lie adjacent to the mouth (top left of cell a). Nuclei are not in clear focus.
Two different responses to pseudopodial fusion were exhibited by cell a. Contact
and pseudopod fusion with cell b (Fig. i A, left arrow) was immediately followed by a
withdrawal response. Within 3 s (Fig. 1 B), both parts of the shared pseudopod had
detached from the agar and formed a straight connecting pseudopod. Some irregular
beading followed, and the pseudopods parted without rebound about 9 s after the first
contact. In contrast, pseudopodial fusion between cells a and c (Fig. 1 A, right arrow)
was not followed by rapid withdrawal and remained for nearly 60 s, apparently parting
only as part of the normal extension-withdrawal cycle. Cells a and d did not fuse,
since only the cell mantles touched. Cell d exhibits 2 pseudopodial complexes, one
extending (left), one withdrawing (right); the left pseudopod exhibits slight proximal
shortening with movement of the cell body about 05 fim to the left between A and B.
(Illustrations: AFIP Negs. 69-7644-1-8) x 1350.
Figs. 2-4. Phase-contrast pictures, focused on the pseudopods, of cells moving on a
glass slide. Alphabetic sequences were photographed at 5-s intervals.
Fig. 2A-D. In A and B the upper pseudopod is extending and is preceded by a
flattened, somewhat irregular, adhesive region. In c and D this pseudopod is fully
extended, straight, somewhat thicker near the tip, and lacks any obvious adhesive
region. In the bottom pseudopod, minute adhesive fans are present at the tip in A and
B. In c the distal part is detached and folded upward and continues shortening in D,
while a secondary adhesion at the bend is maintained. During the sequence A-D another
pseudopodial complex advanced toward the right, just above the centre of the cell.
New extensions were relatively narrow and preceded by formation of small adhesive
fans. No pseudopods extended into the medium above the slide. Proximal shortening
can be measured in sequence B-D in the pseudopod complex to the right. The base of
the branch pseudopod is stationary with reference to bacteria adhering to the slide.
The cell body is about 6 fim from this branch in B and about 3 /tm from it in D,
10 s later, x 750.
Fig. 3 A—D. The cell and field of view are the same as in Fig. 2. Fig. 3 A was 30 s
later than Fig. 2D. During the interval, the upper pseudopod retracted by about half
its length and re-extended to the left of its position in Fig. 2. In Fig. 3 A the upper
pseudopod is in the hold position. Five seconds later (B) small lateral adhesions
formed on this pseudopod to become, within 5 s (c), a branch pseudopod; 5 s later (D)
the 2 had fused and retraction had started. The lower right pseudopodial complex in
Fig. 3 was derived from the centre right complex in Fig. 2. In 3A-C the lower branch
is extending, led by a minute adhesive flattening. In c 2 pseudopodial branches of this
complex were fused, and in D all 3 had fused in a zipper-like action, prior to retraction.
X7S°Fig. 4A-C. Bacteria adhering to the substratum form reference points for 3 fused
cells moving by means of shared pseudopods. Features noted in Figs. 2 and 3 can also
be seen in shared pseudopods in this sequence. The larger amount of cytoplasm
available to multiple cell clusters permits more extensive protoplasmic sheets, which
form and retract between pseudopods (top right). The 4 bacteria (2 at arrows) within
the pseudopods were initially attached to the glass and were covered and detached
as pseudopods extended. A bacterium, just contacted in Fig. 4A (arrow), was covered
and detached within 5 s (Fig. 4B). The bacteria were carried under the cell bodies as
pseudopods retracted. Two single pseudopods pointing toward the upper left and
bottom right can be seen in Fig. 4A; within 5 s (B) both these pseudopods had
withdrawn, x 750.
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J. L. Griffin
Fig. 5. Electron micrograph of parts of 3 cells that share a pseudopod (p). Each cell is
covered by a mantle that joins an oral collar (oc) at the mouth. Within the cell bodies
are seen mitochondria (m), endoplasmic reticulum (er), food vacuoles(/t>), cytoplasmic
bacteria (6), contractile vacuoles (cv), and a nucleus («) with a complex nucleolus.
Glutaraldehyde. x 10000.
Figs. 6, 7. Enlargements of the mouths of the left and upper cells in Fig. 5. One side
of the oral collar (oc) is directly connected to the mantle (mri), while the other is
anchored to the plasma membrane at a region of increased density (arrow). In the
pseudopod (p) within the mouth are 16—18 nm microtubules (mt). No microtubules
were seen in the shared pseudopod outside the mouth. Vesicular material within the
emerging pseudopods is seen in tangential section to be in close contact with microtubules. Glutaraldehyde. x 3Z000.
Movement and fine structure of an enclosed amoeba
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Fig. 8. In this longitudinal section a pseudopod emerges from the mouth and diverges
up and down. A parallel array of 20—22 run microtubules (mt) lies within the
pseudopod (p). Above and below the mouth are regions of apparent pseudopod-mantle
adhesion (between arrows) associated with microfilamentous densities (ntf) in the
pseudopod. The wall of the bacterium (b) can be resolved into 5 layers. Apparent
debris (deb) lies between the mantle and the plasma membrane, oc, oral collar.
Osmium tetroxide. x 50000.
Movement and fine structure of an enclosed amoeba
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Fig. 9. In this longitudinal section through the mouth, 18-22 nm microtubules (mi)
extend into the cytoplasm from the junction of the oral collar and the cell membrane.
Microfilaments (mf) are concentrated along the right side of the pseudopod, where the
pseudopod is adjacent to and apparently adheres to the agar. Increased membrane
density and associated microfilaments are present in an apparent pinocytotic
invagination (pi). Dense microfilaments (arrow) in the upper part of the pseudopod
seem to be adjacent to a site of pseudopod-mantle adhesion. A mitochondrion (in)
lies just outside the mouth within the pseudopod. Osmium tetroxide. x 38000.
Figs. 10-13. Micrographs printed at the same magnification to show trilaminar unit
membranes of Difflugiella and Eiiglyplia.
Fig. 10 shows pseudopod membrane (9-10 nm) of Difflugiella fixed with glutaraldehyde and stained in block with uranyl acetate. This special stain reveals the trilaminar
structure but the membrane is not as thick or as easily seen as the pseudopod
membrane (12-13 nm), osmium fixed, of Euglyplia in Fig. 11. Fig. 12 shows the
membrane (9-10 nm) of Euglypha inside the shell (shell visible at top), fixed with
osmium, compared with the membrane of Difflugiella inside the mantle, Fig. 13, also
fixed with osmium. In Fig. 13, note the pale appearance of the Difflugiella membrane
(centre, 8-10 nm) as compared with the myelin figure membrane (top, 9-11 nm),
apparently produced by digestion of bacteria and excreted beneath the mantle,
x 114000.
Movement and fine structure of an enclosed amoeba
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Movement and fine structure of an enclosed amoeba
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