J. Cell Sci. 2, 465-472 (1967)
Printed in Great Britain
465
CYTOPLASMIC STREAMING AND
MICROTUBULES IN THE COENOCYTIC
MARINE ALGA, CAULERPA PROLIFERA
D. D. SABNIS AND W. P. JACOBS
Biology Department, Princeton University, Princeton, N.J., U.S.A.
SUMMARY
Two distinct patterns of cytoplasmic streaming in the leaf of Caulerpa prolifera are described.
Broad, longitudinally running, two-way streams are restricted to the endoplasm of one leaf
surface. Also present are large numbers of narrow, two-way streams that coil helically throughout the endoplasm surrounding the central vacuole. Numerous unique bundles of aggregated,
evenly spaced, oriented microtubules are distributed within the inner cytoplasm some distance
from the cell wall. Cortical microtubules, as described for other plant material, have been only
very infrequently encountered in Caulerpa and appear to be sparsely distributed. Apart from
the prominent bundles of oriented microtubules, no other significant ultrastructural differences
were noted between the stationary ectoplasm and streaming endoplasm. The possible cytoskeletal role of the oriented microtubules in the development and maintenance of asymmetries
in organ differentiation is discussed in relation to their direct or indirect influence on the
directional migration of cytoplasmic components.
INTRODUCTION
Although there have been numerous reports of the occurrence of microtubular and
microfibrillar elements in the cytoplasm of a variety of cell types, only a limited
number of publications has described these structures in algal cells (Berkaloff, 1966;
Nagai & Rebhun, 1966). The possible functions of cytoplasmic microtubules and
microfilaments in the plant cell have been the subjects of some considerable conjecture and controversy. Microtubules have been considered to play a role in the
laying down of secondary walls in differentiating tissue (Wooding & Northcote, 1964),
and cell-plate formation in dividing cells (Pickett-Heaps & Northcote, 1966). Proponents of their possible role in protoplasmic streaming have noted their frequent
occurrence in the regions of the cytoplasm where vigorous streaming occurs, and
their orientation in the direction of streaming (Ledbetter & Porter, 1963, 1964). It has
been suggested that they serve a cytoskeletal function, providing a framework along
which the motive force for streaming may be generated (Cronshaw, 1965 a). This
framework would also serve to orient the flow and deposition of the precursor molecules required for the synthesis of secondary cell-wall layers. O'Brien & Thimann
(1966) have suggested that cytoplasmic microtubules and microfilaments may both be
functional in streaming and may arise from one another in the cell.
There has been no report dealing with the ultrastructure of coenocytic algae. The
complex morphological development of Caulerpa makes it an ideal organism for such
studies. Early reports describe its anatomy as revealed by light microscopy (Dostal,
30
Cell Sci. 2
466
D. D. Sabnis and W. P. Jacobs
1929 a; Janse, 1890). Its cytoplasmic streaming in relation to morphogenesis and
regeneration has also been subjected to some investigation and speculation (Dostal,
19296; Janse, 1904). This report is concerned with observed characteristics of
streaming in this organism and a possible association of microtubules with this
phenomenon. For a morphological description of C. prolifera the reader is referred to
Fritsch (1935), Dostal (1945) or Jacobs (1964).
MATERIAL AND METHODS
C. prolifera (Forsskal) Lamouroux was obtained from the coastal waters of Key
Largo, Florida, and cultured in this laboratory. The algae were grown in synthetic sea
water supplemented with dibasic sodium phosphate (0-02 g/1), sodium nitrate (o-1 g/1)
and soil extract. The temperature was maintained at25 °Cand the lightcycleat i2-i2h,
the intensity of illumination being about 200 ft-c. At intervals of 3 weeks the algae
were cleaned and transferred to fresh medium.
It was practicable to follow cytoplasmic streaming only in the leaf of the alga, as
this structure is flat enough to be examined under the microscope and the cell wall in
this region is thinner and much less opaque than in the rhizome. It is assumed that the
general pattern and rate of streaming is similar in other regions of the cell. The leaf
was isolated by forming a ' pressure wall' (Jacobs, 1964) at the junction of the leaf and
rhizome, and excising the former. Streaming was followed and timed over various
regions of the leaf by tracing the path of starch grains or chloroplasts under the
microscope with the aid of a micrometer eyepiece and a stop-watch.
For electron microscopy, segments about 2 cm in length were isolated by pressure
walls from various regions of the rhizome and the cylindrical petiolar region of the
leaf. These segments were excised and fixed for 5 min in a refrigerated solution of
6-5 % glutaraldehyde in O-IM cacodylate buffer (pH 7-6) to which calcium (o-oi %
CaCl2) and magnesium (O-OOIM MgCl2) salts were added. From the centre of each
tissue segment, smaller pieces (about 1-5 mm long) were cut and returned to the
glutaraldehyde fixative for 2-3 h at 3 °C. Small slivers from the leaf lamina were cut
directly into the fixative. The tissue was washed for 3 h in o-1 M cacodylate buffer
containing 0-25 M sucrose. Secondary fixation was in cold 1% osmium tetroxide
similarly buffered. The tissue was dehydrated in ethanol or acetone and embedded in
Epon 812. Silver sections were cut with a diamond knife on a Sorvall-MT2 ultramicrotome. The sections were stained with a saturated solution of uranyl acetate in
50 % ethanol (20 min) followed by Reynold's lead citrate (20 min). Grids were
examined in a Hitachi HS-7S electron microscope operating at an accelerating
voltage of 50 kV.
OBSERVATIONS
Cytoplasmic streaming
The leaf of C. prolifera is characterized by a cylindrical petiolar region at the base
that expands into a flat lamina. At the apex, the leaf often has a depression or notch
that sometimes results in a bilobed structure. In young, rapidly expanding leaves, the
Cytoplasmic streaming and microtubules
467
extreme apex is characteristically largely devoid of chloroplasts. The cell wall varies
between 10 and 15 /i in thickness, and below it the parietal cytoplasm consists of a
stationary ectoplasmic layer, about 5-10 [i in depth, and an endoplasmic layer within
which numerous two-way streams are oriented in two distinct patterns. Ovoid
chloroplasts, 3-6 /i long, are present in the ectoplasm and the streaming endoplasm.
Extending from the cell wall into the interior of the cell are numerous wall struts or
trabeculae, possibly with a skeletal function. The cytoplasm also extends over the
surface of the trabeculae and encloses a large central vacuole that extends throughout
the cell. Electron micrographs indicate that the tonoplast is extremely convoluted in
outline, allowing tenuous fingers of the vacuole to penetrate into the cytoplasmic layers.
In the leaf several distinct longitudinally running streams are visible (Fig. 1). These
streams may be as much as 100 /i wide, and the broader streams in the mid-axis may
each contain 20 moving files of chloroplasts across their width. Where the blade is
widest, the outer longitudinal streams tend to diverge towards the leaf edge, forming
a small angle with the long axis of the leaf. This angle is rarely more than 15-18 °.
The 'slanted streams' described in the early literature (Dostal, 1929a) are numerous,
covering the entire surface of the leaf and oriented as in Fig. 2. In the mid-line, the
angle formed with the long axis of the leaf was 45-50 °. These streams are approximately 5-10 [i wide and generally contain only a single file of chloroplasts and starch
grains. Owing to the thickness and opacity of the material, the migration of organelles
could be followed across only one surface of the leaf. However, careful examination of
both leaf surfaces suggest that the 'slanted streams' actually trace a helical course. At
least this form of streaming in Caulerpa is distinctive in contrast to the rotational
cyclosis described in Nitella (Kamiya, 1959; Nagai & Rebhun, 1966) and many other
algae. Towards the edges of the leaves some branching and fusion of streams is
apparent.
The longitudinal streams lie at a level below the spiral streams within the interior
of the cell and apparently flow in the endoplasm underlying only one surface of the leaf.
As the spiral streams are evidently not restricted to one leaf surface, this phenomenon
is a curious one and calls for closer examination. At this stage, any speculation as to
the morphogenetic function of this asymmetry would be pointless.
The direction of movement in the longitudinal streams seems fairly clear as the
streams moving in opposite directions are located at different levels within the cytoplasm. The upper streams (those closer to the leaf surface) flow acropetally, i.e in the
base-to-apex direction, whereas the lower ones move in the reverse direction. The
spiral streams are also located in at least two different adjacent levels within the
endoplasm and movement is bidirectional. However, the narrow streams run so close
together in both the horizontal and vertical axes that it is difficult to decide whether
the pattern in this case also is one of separate cytoplasmic layers streaming in opposite
directions.
The rate of streaming in Caulerpa is relatively slow, varying from 3 to 5 /i/sec, as
compared with 60 /i/sec in Nitella (Kamiya, 1959). As a general rule, it appears to be
more rapid in younger, growing leaves that it is in mature leaves.
30-2
468
D. D. Sabnis and W. P. Jacobs
Cytoplasmic microtubules
Various techniques of fixation were attempted and the methods finally employed
appear to provide the best general preservation of the cytoplasmic contents. Chloroplasts, nuclei, mitochondria and the Golgi complex were well preserved. There was
little vesiculation of the cytoplasm, which abounded in ribosomes, generally aggregated
into polyribosomal clusters. The endoplasmic reticulum was largely rough and enclosed prominent cisternae. The vacuoles contained numerous irregular, electrondense bodies (db) that probably represent a storage product. These disappear unless
the tissue is post-osmicated and may possibly contain a lipid component. None of the
fixation techniques tested could prevent some detachment of the plasma membrane
from the cell wall, but the former generally remained intact. Detailed observations on
the general ultrastructure of this alga will be published elsewhere.
Electron micrographs showed that the cytoplasm was replete with microtubules,
usually aggregated into long bundles or arrays. The presence of such distinct bundles
was particularly prominent in sections of the leaf. Fig. 3 presents a view at low magnification of a leaf sectioned in a plane slightly oblique to the surface of the blade. The
area seen is representative of the leaf endoplasm adjacent to the central vacuole. In this
region, the cytoplasm is extensively penetrated by the vacuole, and restricted to
strands containing the arrays of microtubules and connecting the scattered organelles.
By contrast, in the dense cytoplasm adjoining the cell wall, the organelles are closely
packed. In this cortical region, the chloroplasts tend to be aligned with their long axes
perpendicular to the wall. On the other hand, as seen in Fig. 3, the chloroplasts in the
vicinity of the bundles of microtubules lie parallel to the latter, a feature invariably
observed in our preparations. The bundles of tubules may be traced over a distance
of 20 [i in a section (Fig. 3). A portion of a large bundle is seen in Fig. 4.
The microtubules are approximately 210 A in diameter, the dimensions being very
similar to those reported for similar structures in other plant material (Ledbetter
& Porter, 1963, 1964; Nagai & Rebhun, 1966). The electron-dense wall of the tubule
encloses a less-dense lumen (Figs. 5, 6). The microtubules also run for considerable
distances with little bending, as has been observed before (Burton, 1966; Cronshaw,
1965 a), and which suggests a rigid structure. The microtubules in a bundle are
usually separated by a constant space of about 300-400 A. The presence of helically
arranged subunits in individual tubules is suggested by the cross-banded appearance
(Figs. 4, 8).
These organized structural elements are not restricted to the leaf of Caulerpa but
are also found in the petiolar region and throughout the rhizome extending to the
growing tip (Figs. 5, 6). A striking feature is their occurrence in bundles only in the
internal cytoplasm some distance from the cell wall, and often adjacent to the tonoplast
(Figs. 6, 7). We have, however, very occasionally observed microtubules in association
with and possibly parallel to the plasma membrane (Fig. 9). These structures are not
aggregated into bundles and appear to be sparsely distributed in a single layer adjacent
to the plasmalemma. As a general observation, the endoplasmic bundles appear to lie
more or less parallel to one another (Fig. 8) and run in a direction somewhat oblique
Cytoplasmic streaming and microtubules
469
to the long axis of the leaf and rhizome. However, owing to difficulties encountered
during embedding and sectioning in accurately orienting the fragments of tissue
excised from the cell, we are not yet in a position to relate conclusively the orientation
of the microtubules and the cytoplasmic streams seen in Figs. 1 and 2.
Except for the arrays of microtubules, no other differences were noticed in the
fine structure of the ectoplasm and the endoplasm. No structures corresponding
to the 50-A microfilaments described by Nagai & Rebhun (1966) in Nitella were
observed.
DISCUSSION
In the discussion that follows we shall consider briefly some cellular functions that
have been attributed to microtubules and attempt to justify the suggestion that they
serve a cytoskeletal function in Caulerpa, with a direct or indirect influence on cytoplasmic streaming. A belief that microtubules may be associated with streaming does
not imply accrediting these structures with generating the motive force responsible
for it. Evidence is now accumulating to suggest that within some cells microtubules
may possibly provide the structural framework that directs the orientation of more
than one phenomenon. Since the original suggestion of Ledbetter & Porter (1963,
1964) that microtubules might exert an influence on the disposition of cell-wall
material, a number of publications have pointed out that the orientation of cortical
microtubules mirrors that of the microfibrils in the most recently deposited wall layer
(Cronshaw, 1965 a, b, Cronshaw & Bouck, 1965; Hepler & Newcomb, 1964; Wooding
& Northcote, 1964). On this basis it has been suggested that microtubules might be
involved in the transport of cell-wall precursor material. Cronshaw (1965 a), however,
has pointed out that cortical microtubules are sometimes seen to be attached to the
plasmalemma at both ends, undermining the likelihood that they are functional in a
transporting role. He suggests instead that the oriented skeleton may trap and direct
wall metabolities, in addition to being concerned with either the generation of motive
force or the directing of cytoplasmic streaming. Newcomb & Bonnett (1965) found
that in the young root hairs of radish, the microfibrils of the inner wall layer and the
adjacent microtubules were similarly oriented some distance behind the tip. However,
the oriented microtubules also extend into the zone near the tip where the wall
structure consists of random microfibrils.
It may be mentioned here that the cell walls of Caulerpa are extremely atypical in
that they are composed largely of xylan in which xylose residues are linked by a 1,3-bond
(Iriki, Suzuki, Nisizawa & Miwa, i960). Preston (1962) and Frei & Preston (1964)
have shown that although the xylan walls contain well-defined microfibrils, their
arrangement is on the whole random. The microfibrils within the trabeculae lie banded
together in close arrays parallel to the trabecular axis. However, the outer surface of each
trabeculum is covered with a meshwork of randomly arranged microfibrils. Therefore,
in this organism at least, it is difficult to associate wall deposition with oriented microtubules, even if it were only those adjacent to the plasma membrane that were
involved.
47O
D. D. Sabnis and W. P. Jacobs
What evidence, then, can we muster that may suggest that microtubules are involved
in cytoplasmic streaming? It seems significant that in Caulerpa whereas the cortical
region contains very few microtubules, the inner portion extending up to the tonoplast,
and presumably representing the streaming endoplasm, is filled with prominent
bundles of regularly arranged and distinctively oriented microtubules. Save for these
elements, no other ultrastructural features distinguish ectoplasm from endoplasm.
In relation to the proposed cytoskeletal function of microtubules, their rigidity is
emphasized by the recent observation in the lung-fluke sperm (Burton, 1966), that
increased periods of sonic disruption result in shorter and shorter fragments of microtubules that break transversely and retain their basic structure. In Caulerpa, the
presence of the evenly packed arrays of microtubules might indicate their involvement in a cytoskeletal or supporting role. An aspect of their possible function in the
cell is suggested by the occurrence of homologous structures in relation to pronounced
asymmetries in cell forms (Porter, Ledbetter & Badenhausen, 1964). In their possible
participation in the directional migration of cytoplasmic materials, these structures
may be associated with the development and maintenance of modified cell shapes.
They are found in dividing, differentiating and motile cells. If this were true, then in
a coenocytic cell like Caulerpa, where growth along the longitudinal axis is accompanied
by regularly spaced, timed and oriented differentiation of the rhizome, it would not
be surprising to find a microtubular framework of the proportions described here.
Some similarities between the bundles of microtubules found in Caulerpa and the
similar arrays of microtubules described in the developing oocyte stalk of the freshwater mussel (Beams & Sekhon, 1966) are rather striking. Tilney & Porter (1965) are
also of the view that as cells undergo linear extension, microtubules are often arranged
in the direction of the forming extension. In Caulerpa a pronounced polarity of
development and regeneration exists. It is difficult to visualize a mechanism responsible
for the expression of polarity other than the directed migration of cytoplasmic components. Indeed, studies on regeneration in this organism led earlier authors to propose
this hypothesis more than fifty years ago (Dostdl, 1929a, b; Janse, 1890, 1904).
A last point to relate morphogenesis, microtubules and cytoplasmic streaming is the
observation that surgically-induced diversions in streaming patterns of the leaf effect
profound changes in the subsequent polarity and distribution of organ regenerates.
In conclusion, we believe that there is some evidence, although admittedly indirect,
to suggest that microtubules may serve a cytoskeletal function in cellular differentiation
and serve to provide either the actual framework or to delimit areas of cytoplasmic
substrate upon which the motive force responsible for streaming is generated.
This investigation was supported by funds from a contract between the Office of Naval
Research, Department of the Navy, and Princeton University. The helpful suggestions of
Dr L. I. Rebhun during the course of this work and preparation of the manuscript are gratefully
acknowledged. We are also grateful for the technical assistance of Mr E. Van Norman and the
use of facilities provided by the Whitehall Foundation.
Cytoplasmic streaming and microtubules
471
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Journal of Cell Science, Vol. 2, No. 4
Fig. 1. Surface view of the leaf of Caulerpa showing the longitudinal streams (arrows),
x 10.
Fig. 2. Surface view of the leaf of Caulerpa in the region where the petiole expands into
the flat lamina. The spiral streams are clearly discernible, x 10.
D. D. SABNIS AND W. P. JACOBS
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Journal of Cell Science, Vol. 2, No. 4
Fig. 3. Section through the leaf showing the prominent bundles of microtubules
(arrows) in the cytoplasm adjacent to the central vacuole. Note the parallel orientation
of the chloroplasts lying adjacent to the bundles, (c, Chloroplast; t, trabeculum;
v, vacuole.) x 7500.
D. D. SABNIS AND W. P. JACOBS
Journal of Cell Science, Vol. 2, No. 4
Fig. 4. Section through the leaf showing portion of a large bundle of tubules. The
arrow points to a cross-banded appearance or a periodicity in the substructure of the
microtubules. x 48 000.
Fig. 5. Transverse section of the rhizome. An endoplasmic strand of cytoplasm is
filled with microtubules. x 48000.
D. D. SABNIS AND W. P. JACOBS
Journal of Cell Science, Vol. 2, No. 4
Fig. 6. Transverse section of the rhizome. The tonoplast (t) lining the vacuole (v) is
preserved intact. Numerous microtubules (arrow), sectioned transversely, are seen
concentrated in the cytoplasm bounded by the tonoplast. x 63 000.
Fig. 7. Section through the leaf. Microtubules are again visible adjacent to the
tonoplast, this time sectioned longitudinally, (r, Chloroplast.) X 48 000.
D. D. SABNIS AND W. P. JACOBS
Journal of Cell Science, Vol. 2, No. 4
pm
Fig. 8. Section through the leaf, showing the substructure of the microtubules at a
higher magnification, x 96000.
Fig. 9. Leaf section near the cell wall. Peripheral microtubules (mt) are visible in close
association with the plasmalemma (pm). x 48000.
D. D. SABNIS AND W. P. JACOBS