J. Cell Sci. 2, 465-472 (1967) 465 Printed in Great Britain

CYTOPLASMIC STREAMING AND 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 through- out the endoplasm surrounding the central vacuole. Numerous unique bundles of aggregated, evenly spaced, oriented microtubules are distributed within the inner 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 cyto- skeletal 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 in the have been the subjects of some considerable con- jecture 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). Pro- ponents 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 mole- cules 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 . 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 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 ultra- microtome. 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 approxi- mately 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 cyto- plasm. 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. Chloro- plasts, 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 en- closed prominent cisternae. The vacuoles contained numerous irregular, electron- dense 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 mag- nification 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 cyto- plasmic 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 micro- tubules, 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 micro- tubules that break transversely and retain their basic structure. In Caulerpa, the presence of the evenly packed arrays of microtubules might indicate their involve- ment 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 fresh- water 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 com- ponents. 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

REFERENCES BEAMS, H. W. & SEKHON, S. S. (1966). Electron microscope studies on the oocyte of the fresh- water mussel (Anodonta), with special reference to the stalk and mechanism of yolk deposition. jf. Morph. 119, 477-502. BERKALOFF, C. (1966). Observations sur l'organisation infrastructural d'une Volvocale. C. r. hebd. Se'anc. Acad. Sci., Paris 262, 1232-1234. BURTON, P. R. (1966). Substructure of certain cytoplasmic microtubules: An electron micro- scopic study. Science, N.Y. 154, 903-905. CRONSHAW, J. (1965a). The organization of cytoplasmic components during the phase of cell wall thickening in differentiating cambial derivatives of Acer rubrum. Can. y. Bot. 43, 1401—1416. CRONSHAW, J. (19656). Cytoplasmic fine structure and cell wall development in differentiating xylem elements. In Cellular Ultrastructure of Woody Plants, pp. 99-124. New York: Syracuse University Press. CRONSHAW, J. & BOUCK, G. B. (1965). The fine structure of differentiating xylem elements. y. Cell Biol. 24, 4I5-43I- DOSTAL, R. (1929a) Untersuchungen iiber Protoplasmamobilisation bei Caulerpa prolifera. yb. zviss. Bot. 71, 596-667. DOSTAL, R. (19296). Zur Vitalfarbung und Morphogenese der Meeressiphonen. Protoplasma 5, 168-178. DOSTAL, R. (1945). Morphogenetic studies on Caulerpa prolifera. Bull. int. Acad. Tcheque Sci. 46, T 33-149. FREI, E. & PRESTON, R. D. (1964). Non-cellulosic structural polysaccharides in algal cell walls. I. Xylan in siphonaceous . Proc. R. Soc. B 160, 293-313. FRITSCH, F. E. (1935). The Structure and Reproduction of the Algae, vol. I. Cambridge: University Press. HEPLER, P. K. & NEWCOMB, E. H. (1964). Microtubules and fibrils in the cytoplasm of Coleus cells undergoing secondary wall deposition, y. Cell Biol. 20, 529-533. IRIKI, Y., SUZUKI, T., NISIZAWA, K. & MIWA, T. (i960). Xylan of siphonaceous green algae. Nature, Lond. 187, 82-83. JACOBS, W. P. (1964). Rhizoid production and regeneration of Caulerpa prolifera. Pubbl. Staz. zool. Napoli 34, 185-196. JANSE, J. M. (1890) Die Bewegungen des Protoplasma von Caulerpa prolifera. yb. wiss. Bot. 21, 163-284. JANSE, J. M. (1904). An investigation on polarity and organ formation with Caulerpa prolifera. Proc. Sect. Sci. K. ned. Akad. Wet. 7, 420-435. KAMIYA, N. (1959). Protoplasmic streaming. In Handbuch der Protoplasmaforschung, vol. 8 (ed. L. V. Heilbrunn & F. Weber), p. 3a. Berlin: Springer. LEDBETTER, M. C. & PORTER, K. R. (1963). A ' ' in plant cell fine structure, y. Cell Biol. 19, 239-250. LEDBETTER, M. C. & PORTER, K. R. (1964). Morphology of microtubules of plant cells. Science, N.Y. 144, 872-874- NAGAI, R. & REBHUN, L. I. (1966). Cytoplasmic microfilaments in streaming Nitella cells. y. Ultrastruct. Res. 14, 571-589. NEWCOMB, E. H. & BONNETT, H. T. JR. (1965). Cytoplasmic microtubules and wall microfibril orientation in root hairs of radish, y. Cell Biol. 27, 575-589- O'BRIEN, T. P. & THIMANN, K. V. (1966). Intracellular fibres in oat coleoptile cells and their possible significance in cytoplasmic streaming. Proc. natn. Acad. Sci. U.S.A. 56, 888-894. PICKETT-HEAPS, J. D. & NORTHCOTE, D. H. (1966). Organization of microtubules and endo- plasmic reticulum during and cytokinesis in wheat meristems. y. Cell Sci. 1, 109-120. PORTER, K. R., LEDBETTER, M. C. & BADENHAUSEN, S. (1964). The microtubule in cell fine structure as a constant accompaniment of cytoplasmic movements. Proc. %rd European Regional Conf. Electron Microsc. vol. B, p. 119. PRESTON, R. D. (1962). The microfibrillar structure and coherence of plant cell walls. 5th Int. Conf. Electron Microsc, p. BB-i. New York: Academic Press. 472 D. D. Sabnis and W. P. Jacobs TILNEY, L. G. & PORTER, K. R. (1965). Studies on microtubules in Heliozoa. 1. The fine structure of Actinosphaeriuvi nucleofilum (Barrett) with particular reference to the axial rod structure. Protoplasma 60, 317-344. WOODING, F. B. P. & NORTHCOTE, D. H. (1964). The development of the secondary wall of the xylem in Acer pseudoplatanus. jf. Cell Biol. 23, 327-338.

[Received 17 April 1967) 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 {Facing p. 472) 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, ; 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