J. Cell Sci. 39, 213-232(1978)
Printed in Great Britain © Company of Biologiits Limited 1978
213
ENDOCYTOSIS AND STREAMING OF HIGHLY
GELATED CYTOPLASM ALONGSIDE ROWS OF
ARM-BEARING MICROTUBULES IN THE
CILIATE NASSULA
J.B. TUCKER
Department of Zoology, The University, St Andrews, Fife, Scotland, KY16 gTS
SUMMARY
The microtubular cytopharyngeal basket acts as a jet engine when Nassula ingests filaments
of blue-green algae. Prolonged and highly directed cytoplasmic streaming forms the main
mechanical basis for propulsion of algal filaments through the basket. Cytoplasm surrounding
such filaments streams at the same rate as that at which filaments travel through the basket's
interior. This cytoplasm appears to be very highly gelated. The arm-bearing surfaces of certain
rows of microtubules seem to be located in the active shear zone where the forces responsible
for cytoplasmic streaming are generated.
Algal ingestion represents a rapid and large scale form of endocytosis. An algal filament is
contained in a long membranous invagination from the ciliate's cell surface as it is drawn through
the basket. The invagination moves through the basket at the same velocity as the algal filament
and surrounding cytoplasm. The arm-bearing microtubules do not appear to bind directly to the
invagination which consists of two unit membranes. One of these is probably an invagination of
the cell surface membrane. The origin of the other membrane has not been ascertained.
There are indications that microtubule sliding and a contractile ring of microfilaments
operate antagonistically during feeding to evert and re-invaginate a cell surface depression where
the top of the basket is located.
INTRODUCTION
Transport of a wide range of cytoplasmic particles takes place alongside microtubules in a variety of cell types. For example, ribosomes (Macgregor & Stebbings,
1970), pigment granules (Murphy & Tilney, 1974; Schliwa, 1975) and several types of
vesicles (Hepler & Jackson, 1968; Smith, 1971; Allen, 1974; Gray, 1975) are thus
transported. The exact nature of the contractile elements involved remains to be
established. They are probably anchored to the surfaces of the microtubules in some
instances (Hepler, Mclntosh & Cleland, 1970; Ochs, 1972; Tucker, 1972; Bardele,
1974; Allen, 1975; Smith, Jarlfors & Cameron, 1975), although this does not
appear to be the case for particle movement alongside microtubular axonemes in the
heliozoan Echinosphaerium (Edds, 1975). For most situations it is not yet clear whether
particles are moved through the cytoplasm which immediately surrounds them or if
they are transported because such cytoplasm is streaming alongside the microtubules.
This report shows that the latter situation sometimes obtains.
In several ciliates food materials are propelled alongside rows of arm-bearing
214
J- B. Tucker
microtubules as they are endocytosed at the cytostome and transported through the
cytopharynx (Bardele, 1972; Tucker, 1972, 1974; Hitchen & Butler, 1973; Rudzinska,
1973; Hauser & Van Eys, 1976). Nassula propels portions of blue-green algal filaments
up to 0-5 mm long through its microtubular cytopharyngeal basket (Tucker, 1968) at
rates of up to 30^111 s -1 . This paper deals with the mechanical role of cytoplasmic
streaming and certain arm-bearing rows of cytopharyngeal microtubules when
Nassula is feeding. A previous account of basket activity during algal ingestion was
based on observations of feeding organisms using phase-contrast microscopy (Tucker,
1968). This paper provides much more detailed information which has been obtained
using Nomarski differential interference-contrast microscopy for living organisms and
electron-microscopic examination of thin sections of organisms which were fixed
while feeding.
MATERIALS AND METHODS
Nassula aurea was cultured and prepared for electron microscopy as described previously
(Tucker, 1967). Feeding Nassula were obtained by mixing suspensions of starved organisms in
culture medium with filaments of the blue-green algae Phormidium inundatum and P. unicinatum.
Organisms which were actively ingesting filaments were pipetted individually into the initial
fixation solution for electron microscopy. Ingestion continued while such organisms were in
pipettes and until they came into contact with the fixative. The procedure for obtaining living
feeding organisms on slides beneath coverslips for light-microscopical examination has already
been described (Tucker, 1968). These preparations were examined and photographed using a
Zeiss (Oberkochen Ltd) Universal Microscope fitted with Nomarski differential interferencecontrast optics. Living feeding organisms, and organisms fixed while feeding with a glutaraldehyde fixative (Tucker, 1967), were also examined with this microscope using attachments for
vertical incident lightfluorescenceexcitation. Filters were employed so that light emitted from
a mercury vapour lamp with wavelengths in the range 330-500 nm was used to excite fluorescence and the fluorescent image examined was formed from light with wavelengths greater
than 530 nm.
RESULTS
Basket structure
Basket fine structure has already been described in detail for an unidentified species
of Nassula (Tucker, 1968). The basket of N. aurea is slightly smaller and includes
fewer cytopharyngeal rods, but otherwise its structure is almost identical to that of the
other Nassula species. It performs the same sequence of feeding movements as those
already described (Tucker, 1968). The arrangement of the main basket components is
summarized below. This outline is needed for presentation of new information on the
basket's role during feeding.
The top of a resting basket (one that is not engaged in feeding activities) is attached
to the bottom of a pellicular depression called the oral atrium (Fig. 5A). The cytostome
and a circular collar form the floor of the atrium. The collar consists of a radial array
of thickened corrugations in the pellicular epiplasmic layer. It surrounds the circular
cytostome (Fig. 11, A) where only a single unit membrane separates cytoplasm in the
lumen of the basket from the external environment (Fig. 5A). A circular palisade of
microtubular rods is situated below the collar. The tops of rods are embedded in a
Endocytosis and cytoplasmic streaming
215
fibrous annulus which is largely composed of microfilaments and connects the rods to
the lower surface of the collar (Fig. 15). An annulus of densely staining material
encircles the palisade about 5 fim below its top and the palisade is surrounded by a
microtubular sheath (s) from this level to its bottom (Figs. 15, 16). However, there is
apparently no structural barrier to cytoplasmic movement into and out of the lumen
of the basket at its bottom or through the slits between rods which occur at levels
between the two annuli. A microtubular crest (c) is attached to the outer surface of
each rod (r) at levels above the dense annulus (Fig. 1). Crests follow left-handed
helical paths around the outside of the sheath (Fig. 16) for a further 7/tm below the
dense annulus.
Three types of rows of arm-bearing microtubules (cytopharyngeal lamellae) are
attached to the basket. A rod lamella flanks one side and curves around the inner
surface of each rod along almost its entire length. A cytostomal lamella and a subcytostomal lamella are attached to the inner end of each thickened corrugation of the collar.
In a resting basket each cytostomal lamella projects from the tip of a corrugation to the
centre of the cytostome. Subcytostomal lamella extend downwards into the lumen
of the basket and out through the slits between rods from where they extend for at
least 3 /im into the surrounding cytoplasm. Arms project from the side of each rod
lamella which faces way from the rod to which the lamella is attached. They project
from regions where the walls of adjacent tubules in a lamella appear to make contact
(Fig. 2, arrows). Well defined microtubule arms also project from one side of cytostomal (Fig. 4, arrows) and subcytostomal lamellae. For the latter, this side is the one
which is positioned closely alongside a rod-lamella (x) in a resting basket at levels
where subcytostomal lamellae (y) pass out of the basket lumen through the slits between
rods (Fig. 3). Arms have lengths of about 20 nm and thicknesses of about 7 nm. They
are difficult to detect when longitudinal sections of lamellae are examined and because
of this it has not been established whether they are periodically arranged along the
lengths of tubules.
Eversion of the oral atrium
Before Nassula starts to ingest an algal filament the oral atrium is everted so that the
top of the basket and the cytostome can be brought into contact with the filament.
The circular lip around the atrium's aperture increases in diameter during eversion
and is situated below the top of the basket when eversion is completed (Figs. 5, 6).
Certain cilia which are positioned around the lip are joined to crests (c) by bundles of
microtubules (g) called ciliary connectives (Fig. 10). The tubules in these bundles
apparently overlap with some of those which form the tops of the crests. It is not clear
whether they ramify between crest tubules or just lie along the sides of the crests.
The basal bodies of the cilia are situated about 2 /tm above the tops of crests prior to
eversion. After eversion they are only about 0-4 /«m from the crest tops (Figs. 5, 10).
Hence, the portions of the ciliary connective tubules which run between the basal
bodies and the crest tops have shortened by about 1-5 /on. The tubules themselves do
not appear to shorten. Their longitudinal axes are quite straight before and after
eversion and their external diameters remain constant at about 24 nm. During
216
J.B. Tucker
eversion these tubules apparently slide downwards relative to adjacent crest tubules.
If this sliding is an active process it perhaps provides the contractile force which
effects atrial eversion.
The eversion is maintained throughout the period (up to 4 min) of algal ingestion;
re-invagination occurs when ingestion is completed. Considerable numbers of microfilaments (/) are concentrated in, and follow circular paths around, the inside of the
atrial lip (Fig. 7). This circular band of microfilaments may be contractile and provide
a restoring force to constrict the atrial aperture and re-invaginate the atrium when
ingestion is concluded.
Algal ingestion
After atrial eversion a cytoplasmic extrusion starts to swell out from the top of the
basket. The extrusion pushes the thickened corrugations of the collar upwards; they
pivot on their outer ends and their inner ends splay apart (Figs. 5, 12). The extrusion
bulges around the portion of the algal filament which lies against the top of the basket
so that the extrusion has a bi-lobed appearance (Figs. 5, 13). This situation is maintained for 2-6 s, then the extrusion starts to move down into the lumen of the basket.
Simultaneously the filament begins to bend into the lumen. It is still surrounded by
the 2 lobes of the extrusion as this occurs (Fig. 8). Extrusion and filament move downward at exactly the same rate. The palisade of rods becomes elliptical in cross-section
as the filament bends into the basket lumen (Fig. 14). Its cross-sectional profile
becomes circular again after the acutely bent portion of the filament has passed out of
its bottom. Thereafter the filament is ingested as a double strand (Fig. 21) until the
shorter 'arm' of the bent filament has passed through the basket. The remaining
uningested portion of the filament is drawn in as a single strand (Fig. 24).
Sometimes Nassula abandons its attempt to ingest a filament shortly after the extrusion has bulged around the filament. When this occurs it is evident that the extrusion
Fig. 1. Cross-section through part of a feeding basket cut at a level just below the
bottom of the fibrous annulus. Subcytostomal lamellae (y) follow straight paths from
the lumen of the basket (at the bottom of the micrograph) through slits between rods
(r), and beyond the outer ends of the crests (c). These lamellae are not positioned ao
closely alongside rod lamellae (x) as they are in resting baskets (compare Fig. 3).
Densely staining material occupies spaces between some of the rod tubules at this level.
Cytopharyngeal vesicles (v) are concentrated in the cytoplasm inside, and adjacent to,
the basket. Two mitochondria (w) are also shown, x 25000.
Fig. 2. Part of a rod in cross-section. A rod lamella flanks its left side and curves
around its inner surface. Regularly spaced arms (arrows) project from the lamella and
appear to make contact with part of the membranous invagination (PI). Feeding
basket, x 166000.
Fig. 3. A portion of a subcytostomal lamella (y) in cross-section at a level where it
passes through the slits between rods and closely alongside a rod lamella (.v) which
is cut in oblique longitudinal section. Arms projecting from the lamellae appear to
make contact or interdigitate. Resting basket, x 250000.
Fig. 4. A cytostomal lamella in cross-section. One edge of the lamella is attached to the
cell surface membrane (u). Regularly spaced arms (arrows) project from the lamella.
Feeding basket, x 129000.
Endocytosis and cytoplasmic streaming
%Wf
2i8
J.B. Tucker
adheres tightly to the surface of the filament. As the ciliate starts to swim away the
extrusion remains attached to the filament, stretches slightly, and then pinches off near
the cytostome. The distal portion of the extrusion is left behind stuck to the side of the
filament (Fig. 23).
Cytostome
Cilium
Epiplasm
Ciliary
connective
Rod
Crest
Algal
filament
Cytoplasmic
extrusion
Thickened
corrugation
Cell
membrane
Fig. 5. Diagrammatic median longitudinal sections through the tops of baskets
showing the shapes of the adjacent pellicular regions. For clarity fibrous annuli and
cytopharyngeal lamellae have been omitted, A, resting basket, B, basket at the start
of ingestion. The oral atrium is everted and a cytoplasmic extrusion is bulging around
an algal filament shown in cross-section. Ciliary connectives have slid downwards
alongside the crests so that basal bodies are closer to crest tops than they are in a
resting basket.
As the bent portion of the filament emerges from the bottom of the basket it
follows a straight path until it encounters the pellicle on the aboral side of the ciliate
which deflects it posteriorly. Then the filament slides around beneath the pellicle
circumnavigating the ciliate's interior up to 3 times as it is coiled up (Fig. 21). The
acutely bent portion of the filament has a cap of cytoplasm on the tip of the bend as it
Endocytosis and cytoplasmic streaming
Fig. 6. Median longitudinal section through the top of a basketfixedwhile ingesting a
single strand of an algalfilament.The thickened corrugations (t) of the collar dip down
into the basket lumen, the oral atrium is everted and its circular lip (arrows) is situated
below the collar and the tops of the rods (r). The membranous invagination (m) and part
of the dense annulus (d) are also shown, x 13000.
Fig. 7. Thelip ofanevertedoralatriumincross-section. Section orientation as in Fig. 6.
Cross-sectional profiles of microfilaments (/) fill most of the lip's interior. The pellicular epiplasmic layer is thicker and denser where it forms the side walls of the atrium's
interior than where it covers most of the remainder of the organism's surface. The
abrupt change in epiplasmic composition occurs at the tip of the lip (arrow). Pellicular alveoli (a) cover the epiplasm over most of the organisms' surface but not inside
the atrium. Most of these alveoli disintegrate during fixation, x 50000.
219
220
J. B. Tucker
emerges from the bottom of the basket. The cap consists largely of cytoplasm which
forms the proximal portion of the extrusion and is driven down the basket ahead of the
descending filament. This cap has a higher refractive index than that of the surrounding
cytoplasm; it is stiff and is tightly attached to the algal filament. For example, the
pellicle bulges outwards (short arrow) as the cap (k) strikes it at the end of the filament's
initial passage across the ciliate (Fig. 9) and the cap remains attached to the filament
during, and for at least several seconds after, this collision.
Cytoplasmic streaming
Throughout ingestion a complex pattern of cytoplasmic streaming takes place in and
around the basket. This can be monitored by observing the movements of cytopharyngeal vesicles. Large numbers of these vesicles (v) are concentrated in and
around the basket (Fig. 1). The rate at which the filament moves can also be ascertained because of its cross-striated appearance due to the walls between adjacent
cells (Fig. 23).
There are 3 main streams of cytoplasm: inner doumflow, inner upflow, and outer
upflow. Most of the cytoplasm in the lumen of the basket moves downwards (inner
downflow) at exactly the same rate as the algal filament. This rate varies considerably
(up to at least 30/tms~1)and to some extent is related to the load involved. For example,
Fig. 8. Lateral view of the upper half of a basket at the start of ingestion. The 2 lobes
(arrows) of a cytoplasmic extrusion project from the cytostome and lie on either side of
an algal filament which is bending into the basket's lumen. Living organisms,
interference contrast; x 3800.
Fig. 9. The acutely bent portion of an algal filament and a cap (k) of highly gelated
cytoplasm striking the pellicle on the aboral side of Nasmla shortly after the filament
first emerges from the bottom of the basket. The pellicle is pushed outwards (short
arrow) where the cap makes contact with it. The hairpin-shaped loop of algal filament
lies in a plane at right angles to the plane of the micrograph. The tip of the loop is
situated at the point indicated by the long arrow. Living organisms, interference
contrast; X3100.
Fig. 10. The pellicular epiplasmic layer (e), a basal body (6), and its ciliary connective
(g), are situated close to the top of a crest (c) at the top of a feeding basket after atrial
eversion. x 62 000.
Fig. 11. The thickened corrugations of the collar have a radial arrangment around the
circular cytostome (/1) of a resting basket. In this micrograph and in Figs. 12, 13 the
basket is positioned just below the focal plane photographed, with its longitudinal axis
perpendicular to the plane of the micrograph. Living organism; interference contrast.
X4700.
Fig. 12. End-on view of the thickened corrugations of the collar. They pivot through
nearly 90 0 relative to their resting arrangement (compare Figs. 5, 11) as a cytoplasmic
extrusion starts to bulge out of the cytostome around an algal filament. Living organisms, interference contrast; x 4000.
Fig. 13. The 2 lobes of a cytoplasmic extrusion bulging around an algal filament at
the start of ingestion. Living organisms, interference contrast, x 2760.
Fig. 14. End-on view of the top of a basket as an algal filament starts to bend into the
basket's lumen (compare Fig. 8). The palisade of rods has an elliptical cross-sectional
profile. Living organisms, interference contrast; x 3100.
Endocytosts and cytoplasmic streaming
221
^
*«_
13
CLl
39
J. B. Tucker
222
Inner upflow
Outer upflow
Inner downflow
Fig. 15. Schematic diagram of a basket viewed laterally with a sector removed to
show the paths (arrows) followed by streams of cytoplasm during ingestion of an
algal filament. All lamellae have been stippled; in all cases the longitudinal axes of their
microtubules are oriented parallel to the longitudinal axes of lamellae and to the
directions in which adjacent cytoplasm is streaming. The cytostomal lamellae
(unlabelled) are the short lamellae attached to the collar which have their longitudinal
axes oriented parallel to the sides of the algal filament. Crests have been omitted for
clarity.
Endocytosis and cytoplasmic streaming
223
algal filaments often form tangled masses. The initial stages of feeding usually involve
ingestion of a portion of a filament which projects from a tangle. After this portion has
been ingested the organism draws the remainder of the filament from the tangle.
The rate at which both filament and cytoplasm pass down the basket decreases
markedly as Nassula starts to withdraw a filament from a tangle. Sometimes movement
stops altogether for periods of up to 30 s before resuming. When such arrest occurs
the inner and outer upflows also cease. When the load is increased because of algal
tangling, or because 2 organisms are indulging in a 'tug-of-war' with a single filament,
the basket usually decreases in diameter (Fig. 24).
A tubular layer of cytoplasm (about 2 fira thick) at the periphery of the basket
lumen streams upwards (inner upflow) along the entire length of the basket. Cytoplasm within about 4 /<m of the basket's outer surface streams upwards (outer upflow),
passes into the basket lumen through the slits between rods above the dense annulus,
and joins the inner upflow. Both upflows stream at about the same rate as the inner
downflow, and both are directed downwards at the collar where they contribute cytoplasm to the inner downflow (Fig. 15). Occasionally vesicles at the edge of the inner
downflow pass into the inner upflow. They suddenly move in the opposite direction as
this occurs. Such reversals have often been observed to take place at levels below the
dense annulus where the sheath separates cytoplasm in the lumen from the outer
upflow (Figs. 15, 16). Hence there really is an inner upflow because the vesicles are too
large to pass between sheath tubules. Cytoplasm for the inner upflow is apparently
drawn into the lumen at the bottom of the basket.
Along the entire length of the basket, cytoplasm in the inner downflow appears to
have a flat velocity profile at right angles to the direction in which it is travelling.
Cytoplasm in the inner down and up flows does not exhibit any detectable decrease in
velocity at the tube-shaped interface where the 2flowspass over each other. Possibly
there is some sort of thixotropic interaction at this interface to provide a lubricant and
minimize generation of viscous forces.
Thin sections of cytoplasm in the lumen of feeding baskets do not reveal any very
marked differences in structure or composition compared with that of cytoplasm
elsewhere in the organism. Luminal cytoplasm contains greater concentrations of
cytopharyngeal vesicles, and ribosome-like particles sometimes appear to be more
closely packed together than elsewhere. This is also the case for luminal cytoplasm in
resting baskets.
As the bent portion of the algal filament moves into the basket lumen the thickened
corrugations of the collar and their cytostomal lamellae slope downwards into the
basket lumen (Figs. 6, 15). As a result the longitudinal axes of all the arm-bearing
lamella tubules are oriented parallel to the direction of cytoplasmic streaming in their
immediate vicinity (Fig. 15). Many of the microfilaments in the fibrous annulus
follow circular paths around the annulus, but some of them take spiral routes from the
inner ends of the thickened corrugations of the collar towards the perimeter of the
annulus. Hence it is possible that contractile elements in the annulus pull the corrugations downwards as the algal filament enters the basket and hold them in this configuration while ingestion continues.
15-2
224
J. B. Tucker
At levels where subcytostomal lamellae pass out through the slits between rods in
resting baskets all of these lamellae are positioned so closely alongside rod lamellae
that the arms on the 2 types of lamellae appear to make contact or interdigitate (Fig. 3).
Some of these lamellae are separated by distances of up to 0-3 /tm in the slits of organisms which werefixedduring ingestion(Fig. 1). Throughout ingestion, at the level of the
slits, the longitudinal axes of the subcytostomal lamellae (y) project straight out (Fig. 1),
radially and slightly downwards from the basket as shown in Fig. 15.
The membranous invaginaticn
The agal filament is contained in a membranous invagination (m) consisting of two
unit membranes as it is ingested (Figs. 6, 16, 18). When ingestion is arrested temporarily because of severe algal tangling as described above, the invagination sometimes
starts to swell away from the filament at one or more points. These swellings move
down the basket at exactly the same velocity as the filament and inner downflow when
ingestion is resumed. Hence membrane for the invagination is supplied in the vicinity
of the cytostome. The possibility that one of the membranes is part of the alga has
been eliminated by examining uningested portions of filaments. There is no unit membrane outside the algal cell wall. One of the membranes, most probably the membrane
next to the filament, may be an invagination of the ciliate's cell surface membrane.
The origin of the other membrane is puzzling. It almost certainly has a 'free-edge' in
the cytostomal region because there is no other membrane at the ciliate's cell surface
which could invaginate to contribute to it (Tucker, 1968,1971). The second membrane
was detected at all levels below the inner ends of the down-sloping cytostomal lamellae,
but both membranes of the invagination were extensively fragmented near the
cytostome of organisms fixed during ingestion so that examination of this region did
not clarify the situation any further.
While the filament is moving through the basket the membranous invagination is so
closely applied to the surface of the portion of the filament in the basket that it cannot
be distinguished from the filament surface when living feeding Nassula are examined.
Provided that ingestion is not arrested the invagination does not start to swell away
from a portion of the filament until at least 30 s after it has passed through the basket.
Once this has occurred the invagination is readily distinguished from the filament
surface (Fig. 17). However, electron micrographs indicate that the invagination
Fig. 16. Basket fixed while ingesting a single strand of an algal filament; both are cut
in cross-section. The membranous invagination appears to make contact (arrrows)
with the inner edges of the lamellae (x) of 3 of the rods (/•). The sheath (J) and crests
(c) are also shown, x 9200.
Fig. 17. Part of the membranous invagination (m) is bulging away from the side of a
portion of an algal filament which has been ingested and coiled up beneath the ciliate's
pellicle (p). This portion of the filament has started to buckle and break transversely.
Living organisms; interference contrast, x 3800.
Fig. 18. A portion of the membranous invagination in the lumen of a feeding basket.
Two unit membranes separate cytoplasm (to the left) from the apparently empty space
(to the right) which separates the invagination from the algalfilamentafter preparation
for electron microscopy, x 100 000.
Endocytosis and cytoplasmic streaming
226
J. B. Tucker
is separated from the filament by distances of up to 2 /tm inside the basket
lumen (Figs. 6,16). The invagination often appears to bulge towards, and make contact
with, the inner surfaces of rods and their lamellae (Figs. 2, 16). Such associations
are almost certainly not representative of the situation in living organisms. There
are several indications that the invagination swells away from the filament at some
stage during preparation for electron microscopy (probably fixation). The degree of
swelling often varies at different levels in the same basket; it is most marked near the
cytostome and sometimes is almost completely lacking at levels just below the bottom
Membranous
invagination
Fig. 19. Diagrammatic lateral view of a basket at an early stage during the ingestion
of an algal filament as it appears in a living organism when the plane of focus of a
differential interference-contrast microscope includes the basket's longitudinal axis.
A, ingestion has started close to one end of the algal filament. B, when the bent portion
of thefilamentemerges from the bottom of the basket it straightens elastically, its tip
swings through an arc as indicated by the dotted arrow, and the membranous
invagination is pulled away from one side of the filament.
of the basket (Fig. 20). The membranes of the invagination are often broken at several
points at levels where swelling is particularly extensive. Observation of living organisms
which start to ingest filaments close to one of their ends leaves little doubt that the
invagination is usually closely applied to the surface of a filament while it travels
through the basket. When the acutely bent terminal portion of such a filament passes
out of a basket it suddenly straightens elastically. As this occurs part of the invagination
Endocytosis and cytoplasmic streaming
227
is pulled away from one side of the filament revealing that it is closely applied to the
portion of the filament which is still inside the basket as shown in Fig. 19.
The real swelling of the invagination which occurs several seconds after a filament
portion has passed through the basket is apparently related to initial stages in digestion
and breakdown of the filament. The filaments are autofluorescent. Within one minute
of a filament portion entering the membranous invagination fluorescent material
escapes from it into these swellings (Fig. 21). Cytopharyngeal vesicles are closely
applied to, and appear to fuse with, a portion of the invagination within a few seconds
of its emergence from the bottomof the basket (Fig. 20). These vesicles closely resemble
the primary lysosomes described for other cells (Maggi, 1973; Rudzinska, 1974) and
presumably release digestive enzymes into the invagination. The filament buckles
(Fig. 17) and breaks at several points into successively shorter lengths. Swollen portions of the invagination start to coalesce with each other and/or pinch off from neighbouring swellings within about 4 min of the start of ingestion to form spherical food
vacuoles (Fig. 22).
DISCUSSION
Cytoplasmic gelation and propulsion of algal filaments
The algal filament elastically resists the bending to which it is subjected at the start
of ingestion. If cytoplasm in the extrusion from the cytostome were not highly gelated
and somewhat stiffer than the filament, and if this cytoplasm, the invaginating
membranes, and the filament did not stick tightly together, the filament would not
bend into the basket top as the extrusion starts to move downwards. Cytoplasm
which contributes to the extrusion forms a cap on the tip of the bent portion of the
filament. The cap is still in a highly gelated state and firmly attached to the filament
and membranous invagination after they have passed through the basket and at
least until they strike the pellicle on the aboral side of the ciliate.
Since cytoplasm streams down the basket at the same rate as that at which the algal
filament is transported there is little doubt that the filament and membranous invagination are carried along by the stream. If cytoplasm in the inner downflow has the same
properties as that in the cytoplasmic extrusion (which contributes to the inner downflow at the start of ingestion), it forms a stiff tube which adheres strongly to the membranous invagination. The fact that this tube of cytoplasm has a flat velocity profile at
right angles to the direction of movement is a further indication that this may be the
case.
Microtubule arms and cytoplasmic streaming
If the downflowing cytoplasm has sufficient mechanical strength to act as a vehicle
for transport of the algal filament and membranous invagination which are embedded
at its centre, the actively contractile elements responsible for transport need not be
bound directly to the filament or the membranous invagination. In view of the prolonged, rapid, and highly directional nature of the cytoplasmic streaming it is likely
that the contractile elements are anchored in a polarized fashion to rigid skeletal
components in the immediate vicinity of the streams. Microtubule bundles are often
228
J. B. Tucker
fairly stiff (Tucker, 1968; Ockleford & Tucker, 1973) and at least some microtubules
have a distinct polarity (Allen & Borisy, 1974; Dentler, Granett, Witman & Rosenbaum,
1974). Several investigations indicate that microtubule arms are sometimes involved
in transport of materials alongside microtubules (Helper et al. 1970; Tucker, 1972;
Bardele, 1974; Smith et al. 1975). The dynein arms on A tubules of cilia and flagella
play an active part in a sliding interaction between tubule doublets (Gibbons, 1975).
There are several indications that the arm-bearing surfaces of the microtubular cytopharyngeal lamellae are the active shearing zones for cytoplasmic streaming. The
longitudinal axes of these tubules are all oriented parallel to the directions in which
adjacent cytoplasm streams. The cytostomal lamellae project upwards when cytoplasm
is extruded upwards out of the cytostome at the start of ingestion (J. V. Wellings,
personal communication) because they are attached to the tips of the collar corrugations
which become so oriented at this stage (Fig. 5 B). They project downwards when
cytoplasm in their proximity streams downwards (Fig. 15). The arm-bearing surfaces
of rod and subcytostomal lamellae appear to make contact in resting baskets but at
least some of them are widely separated in feeding baskets. This separation is more
compatible with the suggestion that lamellae actively drive cytoplasm over their
surfaces than it is with the possibility that they are passive baffles which only guide the
streams of cytoplasm. For example, if the latter were the case, one would expect the
subcytostomal lamellae to be pressed against the rods and not to be widely separated
from them. In addition, the streams of cytoplasm might be expected to bend these
lamellae (which are only one tubule thick) to one side or the other. However, the
lamellae follow straight paths where they project through the slits in feeding baskets.
This is reasonable if they are driving cytoplasm in through the slits. They will be held
Fig. 20. Cross-section of a portion of an algal filament fixed a few seconds after it had
been ingested and passed through a basket. Vesicles (u) are clumped around the
membranous invagination (m). x 19000.
Fig. 21. Nassula fixed while ingesting an algal filament as a double strand. The top of
the basket is situated at the position indicated by the long arrow. The autofluorescent
algal filament is coiled up inside the ciliate. Portions of the filament which had been
ingested for periods longer than 30 s when fixation occurred have started to buckle
and fluorescent material fills regions where the membranous invagination has started to
bulge away (short arrows) from these portions of the filament. This Nassula had only
one old fluorescent food vacuole before ingestion commenced. Fluorescence microscopy, x 6CXJ.
Fig. 22. A length of ingested algal filament with large swellings of the membranous invagination at either end is shown in the centre of the micrograph. Food vacuoles
and portions of disintegrating algal filaments in food vacuoles containing fluorescent
materials leached from the filament are also shown. Living organism 3 min after the
start of ingestion; fluorescence microscopy, x 700.
Fig. 23. Part of a cytoplasmic extrusion left attached to the side of an algal filament
after a Nassula ceased its attempts at ingestion and swam away while its extrusion was
bulging around the filament. Living organism, interference contrast; x 3000.
Fig. 24. Lateral view of the baskets of 2 ciliates in the ' tug-of-war' situation which
develops when both ciliates start to ingest the same agal filament. Both baskets are
narrower than resting baskets. The top of the basket towards the bottom of the micrograph is more constricted than that of the other basket. Living organisms, interference contrast, x 2600.
Endocytosts and cytoplasmtc streaming
230
J. B. Tucker
taut due to tension, directed away from their points of attachment to the collar, set up
as a reaction to the forces propelling cytoplasm in the opposite direction.
Whether the microtubule arms on lamellae might represent all, or part, of the
contractile elements promoting streaming, or are simply anchor points for such
elements, remains to be ascertained. The possibility that arms may be related to
dynein, or interact with actin and/or myosin has been considered elsewhere (Tucker,
1974; Hauser & Van Eys, 1976).
Hydraulic design of baskets
If the arm-bearing surfaces of lamella tubules are the sites of active shearing for
cytoplasmic streaming it follows that the inner downflow, which is closely associated
with only the relatively short (approximately 1-5 /tm long) cytostomal lamellae,
probably represents a stream of cytoplasm escaping from a region of high hydrostatic
pressure at the top of the basket. Such a region may be produced if the rod and subcytostomal lamellae propel cytoplasm upwards (inner and outer upflows, respectively)
and compress it against the undersurface of the down-sloping collar. The latter may
deflect these streams of cytoplasm into the inner downflow (the collar projects upwards
when cytoplasm is extruded upwards past it, and out of the cytostome at the start of
ingestion).
Downward propulsion of all the cytoplasm in the basket lumen would appear to be
a more direct procedure than the bi-directional flushing action described above.
However, if rod lamellae propelled cytoplasm downwards their action would probably
interfere with the outer upflow alongside subcytostomal lamellae where the upflow
enters the lumen through the slits at the top of the basket.
It is not clear why the basket becomes constricted when there is an increased load to
be overcome in order to propel an algal filament through the basket. Constriction
might be brought about by contraction of the fibrous annulus, or an active sliding
interaction between sheath and crest tubules which follow helical paths around the
outside of the palisade of rods (Tucker, 1968).
Comparison with suctorian tentacles
The axonemes of suctorian tentacles are lined with rows of arm-bearing microtubules; a membranous imagination containing food material is drawn down from a
tentacle tip during feeding (Bardele, 1972, 1974; Rudzinska, 1973; Hitchen & Butler,
1973, 1974; Tucker, 1974; Tucker & Mackie, 1975; Curry & Butler, 1976; Hauser &
Van Eys, 1976). A thin tube-shaped layer of tentacular cytoplasm may be propelled
down the inside of the circular array of tubule rows and carry the membranous
invagination with it; cytoplasm which simultaneously streams up the tentacle outside
the axoneme may provide cytoplasm for this downflow (Tucker, 1974). It has been
suggested that the microtubule arms bind to the invagination and that this is an
important feature of the mechanism which propels the invagination down the tentacle
(Bardele, 1972, 1974). However, if the invagination swells as it does in Nassula
during preparation for electron microscopy, the contacts between arms and the
invagination which are apparent in sections of feeding tentacles may be preparative
artifacts (Tucker, 1974).
Endocytosis and cytoplasmic streaming
231
It is most unlikely that lamella arms bind to the membranous invagination in
Nassula. The planes of the cytostomal and subcytostomal lamellae lie at right angles to,
rather than at a tangent to, the portion of the invaginating membrane closest to them,
so that the arms do not contact the membrane. Contact between the arms on rod
lamellae and the invagination would be more hindrance than help because cytoplasm
alongside rods is streaming in the opposite direction to that in which the invagination
is travelling. Furthermore, since the membranous invagination is closely applied to the
sides of the portion of the algal filament inside the basket of a living Nassula, the
invagination surrounding a single algal strand can make contact with the arms on only
about 6 microtubules/lamella flanking the inner surfaces of 4 rods at most. Hence,
contact with a maximum of 24 arm-bearing tubules could be effected. This represents
about 1-5 % of the total number of rod lamella tubules (approximately 1600). Most of
these tubules flank the sides of rods where they are completely inaccessible for
contacts with the membranous invagination. It seems most unlikely that the basket
would be constructed in this fashion if tubule-arms need to bind to the invagination in
order to function.
Shape changes at the top of the basket
The algal filament is apparently pulled against the top of the basket while it is being
bent into it at the start of ingestion. The filament presumably presses the tops of rods
outwards where it crosses 2 diametrically opposite portions of the basket top as this
occurs, and deforms the basket top so that it has the elliptical cross-sectional profile
described above. Previously (Tucker, 1968), it was sugested that contraction of certain
crests in response to stimuli transmitted from cilia around the oral atrium might be
responsible. The mechanism outlined above is more plausible. Some of these cilia are
structurally modified (Tucker, 1971). The suggestion that they act as sensory receptors
and stimulate contractile activity in the crests is still a reasonable one. Because of their
positions, these cilia are the first structures in the oral region to contact algal filaments.
Furthermore, a sliding interaction between the crest and ciliary connective tubules
apparently occurs during atrial eversion which is the initial step in the sequence of
events associated with ingestion. Contact between cilia and the alga may trigger the
start of this sequence.
I thank Mrs Judith Pattisson for skilful assistance and Dr D. H. Lynn for critically reading
the manuscript. Grants from the Science Research Council (U.K.) are gratefully acknowledged.
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(Received 24 May 1977)