J. Cell Sci. 83, 23-35 (1986)
23
Printed in Great Britain © The Company of Biologists Limited 1986
TWO-DIRECTIONAL PATTERN OF MOVEMENTS ON
THE CELL SURFACE OF AMOEBA PROTEUS
ANDRZEJ GREBECKI
Department of Cell Biology, Nertcki Institute of Experimental Biology, ul. Pasteura 3,
02-093 Warsaw, Poland
SUMMARY
Particles of latex, glass and precipitated Alcian Blue were studied cinematographically on the
surface of migrating Amoeba proteus and in the surrounding medium. The majority of the attached
and all unattached particles flow steadily forward in the direction of the endoplasmic streaming and
cell locomotion. Flow on the surface is faster than in suspension. Some particles stuck on the
membrane move backwards from the frontal region. This retrograde transport is slower than the
anterograde flow, and the rate decreases further when the particles approach cell regions adhering
to the substratum, accurately following the pattern of the withdrawal of ectoplasm in the same
zone. Both movements coexist in the same region and retrograde particles may pass anterograde
ones at a distance less than their diameter. Transition from forward flow to backward transport
occurs just behind the frontal cap, where the new ectoplasm is formed. The anterograde movement
is interpreted as reflecting the general forward flow of the laterally mobile fluid membrane
components, which become added to the frontal surface of the locomoting cell; the retrograde
movement as retraction of membrane components that, externally, are linked to the transported
material and, on the cytoplasmic side, to the contractile microfilamentous layer, as is postulated for
cap formation in tissue cells.
INTRODUCTION
The movement of particles on the surface of free-living amoebae has for a long
time been considered as an indicator of the behaviour of the cell membrane during
locomotion. All reports agree that such markers move generally forwards with
respect to the cell, but nevertheless accumulate in the tail region. Stockem (1966)
explained this paradox by stressing that forward transport of markers along lateral
pseudopodia does not coincide with the principal axis of cell locomotion.
The authors of the classical concepts of amoeboid movement usually considered
the membrane of amoebae as a quasi-permanent structure, which must be carried
along as a whole with the moving cell (Jennings, 1904; Mast, 1926; Griffin & Allen,
1959). They invoked some kind of rolling as its mode of transport. Goldacre (1961)
objected to the view that markers reflect the movement of the membrane, attributing
their transport to the electrophoretic forces generated between the poles of the cell.
He claimed that during locomotion the membrane remains stationary and is recirculated through the cell interior, being constantly ingested at the tail and reconstructed at the front. This hypothesis was supported by Chapman-Andresen
(1964) and Jahn (1964) for amoebae and by Shaffer (1963, 1965) for the amoeboid
stages of cellular slime moulds. The presumption that membrane internalization at
Key words: Amoeba proteus, amoeboid movement, cell surface, membrane flow.
24
A. Grebecki
the posterior pole and renewal at the front are so rapid that they may be factors, or
even the motor of locomotion, was later adopted in the interpretation of movements
on the surface of fibroblasts (Harris, 1973; Bretscher, 1976, 1984).
In Amoeba proteus we found (Czarska & Grebecki, 1966) that granular markers
move forward three-dimensionally (not by rolling) and that, moreover, a slight
increase of the viscosity of the medium results in the manifestation of extracellular
streamings of fluid, which follow the direction of movement of each pseudopodium.
We advanced the view that the whole membrane is transported by its continuous
unfolding on the surface of advancing pseudopodia and refolding on the contracting
ones and on the tail. The folding-unfolding hypothesis has been later confirmed by
cinematographic and electron-microscope studies (Stockem et al. 1969; Haberey
et al. 1969; Haberey, 1971; Stockem, 1972) and supported by Wolpert (Wolpert &
Gingell, 1968) in combination with his own (Wolpert & O'Neill, 1962; Wolpert,
1965) concept of the membrane flowing forward owing to lateral mobility of the lipid
bilayer.
However, recent observations of ectoplasmic movements during locomotion
(Grebecki, 1984, 1985) revealed that glass-wool hairs intercepted by amoeba during
migration do not move generally forward. They are transported forwards by the
withdrawing tail but backwards by the advancing frontal region, where the withdrawal of ectoplasm is seen along the fountain zone. This means that the hairs are
transported from both body poles centripetally, but finally accumulate in the tail
because the cell as a whole moves faster than its ectoplasmic layer. The resulting
pattern may be compared with the capping, extensively studied in tissue cells (for
reviews, see de Petris, 1977; Weatherbee, 1981; Oliver & Berlin, 1982; Geiger,
1983; Jacobson, 1983; Bourguignon & Bourguignon, 1984) and revealed also in
amoebae (in Naegleria gruberi by King & Preston, 1977; and in Chaos chaos by
Taylor et al. 1980).
It became necessary to re-examine in combined experiments these two apparently
incoherent phenomena: the general forward flow of particles and fluid medium and
the centripetal movement of glass hairs. The interest in them is increased by the
recent profusion of controversial concepts of the mechanism of transport of surface
markers and cross-linked surface receptors by different crawling and capping cells:
the theories of surf-riding (Hewitt, 1979; Berlin & Oliver, 1982), bulk backward flow
of the membrane (Abercrombie et al. 1970; Harris, 1973) or of membrane lipids
(Bretscher, 1976, 1984), and the hauling of the complexed surface receptors by
microfilaments (de Petris & Raff, 1973a,6).
The objective of the present report is to describe the general bidirectional pattern
of the movement of markers on the surface of A. proteus, on the basis of cinematographic records, which will be later subjected to quantitative analysis.
MATERIALS AND METHODS
Cultures of A. proteus were kept in the standard Pringsheim medium and fed twice a week on
Tetrahymenapyriformis. Polytactic and monotactic forms (see Grebecki & Grebecka, 1978, for the
terminology) were taken for experiments on the 2nd or 3rd day after feeding. Their motion was
Bidirectional
cell surface movement
in Amoeba
25
recorded in monochromatic green light (546 nm wavelength), in normal culture medium, on slides
with glass spacers supporting the coverslip at a distance of 0-5 mm. The experiments were run in a
semi-dark room, at 20 (±2)°C.
The following materials were used in suspension and, or, covering the surface of the slides:
(1) Dow latex beads 1-009, 0-794 and 0-460/an in diameter, diluted 1:1000 (which produced
0-01% dry weight suspension according to the specification of the manufacturer); (2) crushed
glass-wool hairs, 5-15^m in diameter and 15-250^m in length; (3) ground glass powder with
particle sizes ranging from 1 to lO/mi; (4) Alcian Blue precipitate produced within 2-3 days in
0-04 % solution of the dye in the Pringsheim medium enriched with Ca 2+ up to 2 mM, collected by
sedimentation and resuspended in the culture medium (the supernatant did not stain the surface
coat and failed to provoke any motor or endocytotic response by amoebae). All these particles were
used either alone or in different combinations. Some other materials, such as carmine and Indian
ink particles or calcium oxalate and inulin crystals, were occasionally used to repeat experiments
reported by earlier authors.
A Biolar microscope equipped with a standard TV camera and differential interference optics of
the Pluta system (made by PZO, Warsaw) were used with 20x/0-40 and 40X/0-65 planachromatic
objectives. The image was recorded, either directly from the microscope or from the TV screen, by
a Bolex H16 Reflex camera run at 4 or 1 frames per second by a combined Bolex and Robot timelapse equipment. The films were reviewed with an LW International Mark IV Photo-optical Data
Analyzer.
All the pictures shown in Figs 1-8 are prints of selected film frames, keeping stable reference
marks outside the moving cells in a constant position.
RESULTS
Different locomotory forms of A. proteus were filmed in media containing
0-2—0*4% methylcellulose in which minute crystals of inulin or calcium oxalate,
carmine or Indian ink particles and latex beads, were suspended. The cinematographic records confirmed the earlier results (Czarska & Grebecki, 1966),
demonstrating the three-dimensional forward flow of the viscous medium, parallel to
the motor axis of amoeba. Further experiments proved, however, that an increase of
viscosity is not necessary for the manifestation of this phenomenon. As shown in
Fig. 1, latex beads suspended in the standard culture solution also regularly flow
forward in the direction of cell locomotion, not only in contact with the cell surface
but also at a distance of several micrometres from it. The exact transverse velocity
profiles of suspensions flowing around moving amoebae have not yet been studied,
but the present results clearly demonstrate that the latex particles adjoining the cell
surface move forward considerably faster than those freely suspended in the medium
(Fig. 1). It may also be easily estimated that the velocity generally decreases with
distance from the cell (Fig. 4).
The film records demonstrated, moreover, that the extracellular flow is strictly
dependent on the direction and velocity of endoplasmic streaming (i.e. on cell
locomotion). It accelerates, slows down, stops or reverses its direction simultaneously with the endoplasm, when its flow changes either spontaneously or due to
local stimulation of pseudopodial tips by white light or shade. The oscillating
streaming of endoplasm provoked by 0-2% ethanol is also reflected by the same
oscillations in the extracellular flow of latex suspension.
The particles surrounding the cell or contiguous to its surface move forward with
respect to external reference points, as well as to the cell itself, and thus they
A. Grebecki
Bidirectional cell surface movement in Amoeba
27
gradually approach the frontal zone. However, in the frontal pseudopodia of polytactic amoebae they never reach the extremity, but continue to move at a certain
distance from the tip of the pseudopodium, until it is withdrawn. In monotactic
amoebae, on the other hand, the extracellular particles overtake the advancing front
and, as a result, retrograde countercurrents arise.
The apparently contradictory results obtained with glass-wool hairs (Grebecki,
1984, 1985), which move back from the front along the fountain zone, were again
obtained in the present work. Film analysis of the backward movement of glasspowder particles, ranging from 1 to 10 [im in diameter, proved that their varying
behaviour does not depend directly on their size, but rather on the nature of contact
with the cell surface. Only glass particles stuck to the surface move backwards,
whereas the others follow the general forward flow described before. Under a
sufficiently large fragment of glass, as shown in Fig. 2, the development of an
adhesive knob is observed, after which the particle starts to move back.
The backward movement from the front along the fountain zone is particularly
well shown in suspensions of Alcian Blue precipitated by Ca2+ (Fig. 3). Ruthenium
Red precipitates provide similar results. Clusters of those precipitates also adhere to
specialized knobs (Fig. 4). The backward transport of glass particles and Alcian Blue
precipitate is observed in both polytactic specimens (Fig. 2) and in monotactic ones
(Fig. 3), and has the same features in both forms of amoebae.
The general forward flow of particles and the retrograde transport of some of them
in close contact with the cell surface are not two alternative responses that exclude
one another, but may coexist. Fig. 4 shows an aggregate of Alcian Blue moving
backwards against the forward flow of the surrounding latex suspension. The same
pattern of two simultaneous opposite movements has also been recorded in mixtures
of glass powder with latex and in pure suspensions of glass powder or precipitated
Alcian Blue. Even the latex beads closely adjoining the cell surface form, in fact, two
populations. Most of them move forwards as described before, but a few are
transported backwards. Two latex beads when moving in opposite directions, as
those shown in Fig. 5, may cross each other's path at very short distances, which may
be less than their diameter (<1 /xm). Such measurements are possible when the two
opposite movements are recorded on the upper surface of an amoeba.
Fig. 1. Forwardflowof latex beads around polytactic amoeba, on the cell surface (white
arrowheads) and several ;um from it (black arrowheads), demonstrated by fivefilmframes
(A-E) selected at 4-s time intervals. Note that the movement on the surface is considerably faster than in suspension. Large white arrows inside the cell indicate (in Figs 1-8)
the frontal direction of endoplasmic streaming. Bars (Figs 1-2, 4-9), 10f<m.
Fig. 2. A crumb of glass (black arrowhead) intercepted by the tip of a polytactic
pseudopodium (A), rearrangement of its position just behind the frontal cap (B-C) and
its further transport backwards along the pseudopodium, which continues to advance
(C-E). Note that in C-E the particle is kept by an adhesive knob; 10-s intervals between
the successive stages.
Fig. 3. Interception of a clump of Alcian Blue precipitate (black arrowhead) by the front
of monotactic amoeba (A) and its transport backward upon the cell surface (B—C).
Pictures taken at 24-s intervals. Bar, 100 ^m.
Fig. 4. Three stages of the backward transport of a clump of precipitated Alcian Blue
(black arrowhead) with the simultaneous forward flow of latex beads suspended in the
surrounding medium. Latex is shown as two constellations of particles moving generally
forwards but changing their configurations because the velocity decreases with distance
from the cell surface. Note the adhesive knob under the clump of Alcian Blue; 10-s
intervals between the pictures.
Fig. 5. Two neighbouring latex beads on the cell surface moving in opposite directions,
forward (black arrowheads) and backward (white arrowheads), shown in A-D at 2-s
intervals.
Fig. 6. Latex particle on the cell surface (white arrowhead) transported back from the
front of amoeba strictly in unison with the ectoplasmic granules and the basis of a lateral
pseudopodium (black arrowheads), which are withdrawn in the same direction. Pictures
taken at 2-s intervals.
Fig. 7. Forward movement of a group of latex beads on the cell surface (white arrowheads), independent of the backward movement of ectoplasmic inclusions (black arrowheads), shown in the frontal part of a monotactic amoeba at intervals of 4 s.
Bidirectional cell surface movement in Amoeba
29
The most striking feature of the backward movement of particles on the surface of
an amoeba is its precise coordination with the movement of ectoplasmic inclusions on
the opposite, cytoplasmic side of the cell membrane, which is observable with all
suspended materials applied in this study. The extracellular retrograde transport is
from the beginning slower than the extracellular forward flow; it gradually slows
down further and eventually stops at the middle body region, where the cell is firmly
attached to the substratum and the ectoplasm comes to a halt. The backward
movement of extracellular particles is detected only in those cell regions and
situations that favour the withdrawal of ectoplasm in the form of the fountain
movement. The coordination of movements on both sides of the cell membrane is
particularly easy to record when latex is used as marker. Fig. 6 demonstrates that a
latex bead transported backwards does not change its position with respect to the
underlying ectoplasmic inclusions, but moves strictly together with them. Extracellular particles moving in a retrograde direction also keep a constant position with
respect to small lateral pseudopodia, hyaline blebs and other elements of the cell
contour, which also move backwards together with the withdrawing frontal segment
of the principal ectoplasmic cylinder. Fig. 7 shows that, in contrast, latex moving
forwards in the same cell region, though apparently in contact with the cell surface,
does so independently of the behaviour of the ectoplasmic layer.
In polytactic amoebae particles are usually transported backwards if they are
picked up by the frontal tip of a pseudopodium during its progression (Fig. 2). This
also occurs in monotactic forms to those particles that reach the front after a phase of
flowing forwards (Fig. 8). Their forward movement stops usually just behind the
frontal cap, in the zone where new ectoplasm is being formed from the endoplasm.
There the halted particle may be repositioned, often being rotated, and then
transported backwards in unison with the ectoplasmic layer. The change of direction
of the glass particle in the endoplasm-ectoplasm conversion zone of a monotactic
amoeba is shown in Fig. 8. Many examples of exactly the same behaviour were
recorded with different kinds of particles, including latex beads.
The retrograde ectoplasm-dependent transport of a particle may cease at any
moment and then it immediately joins the others that flow over the cell surface
forwards. Otherwise, the withdrawing particles reach the region where the ectoplasm
is stationary, stop there, are gradually incorporated into the tail part of the amoeba
and eventually shed into the medium. It seems relevant that sometimes the
ectoplasm-dependent backward transport of a latex bead down to the tail of a
monotactic cell may be terminated by its endocytosis. As demonstrated in Fig. 9,
the bead is engulfed by hyaline micropseudopodia developed in the constriction
preceding the uroid, that is close to the zone that is also known (WohlfarthBottermann & Stockem, 1966) to be a site of permanent pinocytosis in amoebae.
DISCUSSION
It may be concluded that the behaviour of foreign particles around and upon the
cell surface of a migrating amoeba is composed of their anterograde and retrograde
30
A. Grebecki
Bidirectional cell surface movement in Amoeba
31
transport, which are two distinct types of movement driven by different mechanisms, though they can coexist in the same cell regions and at the same time. In spite
of their differences, both movements seem to be generated locally and everywhere
along the cell surface, and thus reflect the intrinsic movements of two different
fractions of the surface material. As far as the retrograde component is concerned,
this conclusion is strongly supported by strict coupling of the movements of
extracellular particles and ectoplasmic granules on the two opposite sides of the
plasma membrane. It seems less obvious in the case of the anterograde transport of
particles, especially those lacking direct contact with the cell. It might be supposed
that they are pulled, owing to the viscosity of the medium, by the advancing front of
the amoeba, or driven by any force developed at one or both body poles (as in the
electrophoretic hypothesis of Goldacre, 1961). However, any movements not generated locally would be forcibly hampered close to the membrane by friction. The fact
that the highest speed of particles occurs in contact with the membrane and their
velocity declines with distance from the membrane indicates that the driving force is
generated everywhere locally by the cell surface, which itself moves forward.
Coexistence of two opposite movements within the cell membrane (and the surface
coat) of amoeba is fully*conceivable in view of the lateral fluidity of membrane lipids,
postulated long ago for amoeboid cells by Wolpert (1965), and now commonly
accepted as a part of the fluid mosaic model of membrane structure and function
(Singer & Nicolson, 1972). The fact revealed by the present experiments, that two
latex beads moving in opposite directions may cross each other at a distance less than
1 ;Um, clearly demonstrates the high fluidity of the cell surface material in A. proteus.
Probably the membrane lipids, with those proteins that are not anchored to
cortical microfilaments, flow forwards, pulled by the advancing front due to the
internal 'microviscosity' of the lipid bilayer. That seems to be the only mechanism
capable of accounting for the demonstrated correlation between endoplasmic streaming and the extracellular anterograde flow. The stock of material ready to flow
forwards is perpetually liberated from the posterior surface areas by their gradual unfolding and regenerated by refolding the surface of retracted pseudopodia
(Czarska & Grebecki, 1966).
The function of the general forward flow of the cell membrane in a moving amoeba
is to supplement the surface material in the anterior part of the body as necessitated
by changes in its shape and position. These changes are too fast in amoebae to be
Fig. 8. Transition from the forward flow to the backward transport in monotactic
amoeba. The particle of glass powder (black arrowhead) approaches to the front in A-C,
is held and rotated at the endoplasm-ectoplasm conversion zone in D, and transported
backwards in E—H. Intervals of 10 s separate the successive stages.
Fig. 9. Endocytosis of a latex bead (white arrowhead), which was transported backwards
along the surface of monotactic amoeba up to the basis of the uroid. u and/in A show,
respectively, the uroidal and frontal directions. Note the hyaline micropseudopodium,
which keeps the latex particle in A and retracts with it in B-C. A much larger hyaline
pseudopodium develops in D-E, and engulfs the bead in F by invagination. Irregular
intervals between the successive pictures; A-C stages are repetitive and may last
15-100 s, D-F stages took in the recorded case 6 s.
32
A. Grebecki
compensated for by intracellular membrane turnover, as was demonstrated by
immunofluorescence techniques (Wolpert & O'Neill, 1962) and by the study of the
dynamics of permanent endocytosis (Stockem, 1972). Therefore, the membrane is
recirculated between the tail and the front of locomoting amoeba, at the cell surface.
As to the retrograde component of extracellular movements, it may be postulated
that a particle is transported backwards if the surface receptors responsible for its
adhesion to the cell become (or were) attached to the submembranous layer of
microfilaments. Direct demonstration of such a linkage between a backwardly
moving particle and the cortical ectoplasm is still lacking, but some present observations strongly suggest its existence: (1) the retrograde transport is manifested only
in association with the withdrawal of the ectoplasmic layer in the fountain pattern;
(2) the particles move backwards precisely together with the ectoplasmic inclusions
on the other side of the cell membrane; (3) they move back in synchrony with small
lateral pseudopodia and other elements complicating the cell contour; (4) the
backward movement, contrary to the forward one, is possible only in contact with the
cell surface; (5) adhesive knobs may be seen by light microscopy under the larger
particles during retraction; (6) some retracted particles are eventually endocytosed
by invagination, which is generally recognized as depending on contraction of
cortical microfilaments (Allison & Davies, 1974; Klein & Stockem, 1979). Moreover,
the existence of a link between the activated surface receptors and the contractile
submembranous layer has been demonstrated in other cells, when patches of
receptors cross-linked by lectins, antibodies or other ligands move back to form caps
(Edelman, 1976; de Petris, 1977; Bourguignon & Bourguignon, 1984).
It is proposed in consequence that some particles on the surface of amoeba are kept
there by adhesion, i.e. are attached to the contractile apparatus. Therefore, they
accurately follow all movements of the ectoplasmic cylinder as described here and
before (Grebecki, 1984, 1985), independently of the unfixed fluid surface material,
which flows over them in the frontal direction.
The retrograde transport of particles on the surface of A. proteus presents some
common features with the saltatory motion of particles on the surface of reticulopodia of a foraminiferan Allogromia, though the first seems to depend on microfilaments and the second on microtubules. The latex beads adhering to reticulopodia
also manifest phases of motion accurately coupled with the movement of intracellular
organelles (Bowser et al. 1984); their saltation ceases if the cytoskeleton-membrane
contact is broken, and a link between extracellular particles and the cytoskeleton is
demonstrated in detergent-extracted reticulopodia (Bowser & Rieder, 1985).
Other analogies appear in respect of the backward movement of foreign particles
on the surface of fibroblasts (Ingram, 1969; Abercrombie et al. 1970; Harris, 1973;
Albrecht-Biihler, 1973) and growing axons (Bray, 1970), as well as of the segregation
of receptor—ligand complexes in the course of capping (cf. de Petris, 1977;
Bourguignon & Bourguignon, 1984). In fibroblasts a retrograde transport of particles
is seen on the surface of a lamellipodium, in front of the firm cell-to-substratum
attachment zone, i.e. where in amoebae the fountain movement may arise; it also
slows down gradually en route from the leading edge to the cell centre; it is also well
Bidirectional cell surface movement in Amoeba
33
correlated with backward motion of the outer cell contour (ruffles and blebs) and
"a generalized backward movement" occasionally seen inside the lamellipodia
(Abercrombie et al. 1970; Harris, 1973).
Nevertheless, the bulk backward membrane flow proposed as the mechanism of
movement of markers upon the surface of fibroblasts by Abercrombie et al. (1970)
and Harris (1973), as well as the backward lipid flow postulated by Bretscher (1976,
1984), are inadequate to explain the present results. In A. proteus a general flow of
the surface material, attributed mainly to its lipid fraction, is evident but directed
forwards. Neither of the surf-riding models (Hewitt, 1979; Berlin & Oliver, 1982)
seems applicable, because of the lack of any system of surface waves in amoeba that
could account for the simultaneous transport of neighbouring particles in two
opposite directions.
On the contrary, the mechanism proposed here for amoeba is fully in line with the
theory put forward to explain the capping of cross-linked receptors by de Petris &
Raff (1973a,b), admitted by Harris (1973) as an "alternative hypothesis", and
adopted by many followers (cf. Bourguignon & Bourguignon, 1984). According to
this theory, patches of receptors are linked to the contractile apparatus and hauled
backwards by it. Moreover, as stressed both by de Petris & Raff (19736) and Harris
(1973), such a model necessitates compensatory countercurrents in the capping cells.
The present work shows that in A. proteus such a countercurrent actually exists in the
form of a general forward surface flow, at least during locomotion.
This study was supported by the Research Project MR.II.l of the Polish Academy of Science.
Film records of the present experiments were shown at the Cell Motility Symposium (Villefranchesur-Mer, 8-12 Sept. 1985) and a poster at the 2nd Polish Conference on Cell Biology (Warsaw,
18-20 Sept. 1985).
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{Received 19 September 1985 -Accepted 19 November 1985)