cytoplasmic streaming and microtubules in the … · marine alga, caulerpa prolifera d. d. sabnis...

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

CYTOPLASMIC STREAMING AND

MICROTUBULES IN THE COENOCYTIC

MARINE ALGA, CAULERPA PROLIFERA

D. D. SABNIS AND W. P. JACOBSBiology 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 leafsurface. 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 cytoplasm some distancefrom the cell wall. Cortical microtubules, as described for other plant material, have been onlyvery infrequently encountered in Caulerpa and appear to be sparsely distributed. Apart fromthe prominent bundles of oriented microtubules, no other significant ultrastructural differenceswere noted between the stationary ectoplasm and streaming endoplasm. The possible cyto-skeletal role of the oriented microtubules in the development and maintenance of asymmetriesin organ differentiation is discussed in relation to their direct or indirect influence on thedirectional migration of cytoplasmic components.

INTRODUCTION

Although there have been numerous reports of the occurrence of microtubular andmicrofibrillar elements in the cytoplasm of a variety of cell types, only a limitednumber of publications has described these structures in algal cells (Berkaloff, 1966;Nagai & Rebhun, 1966). The possible functions of cytoplasmic microtubules andmicrofilaments in the plant cell have been the subjects of some considerable con-jecture and controversy. Microtubules have been considered to play a role in thelaying 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 frequentoccurrence in the regions of the cytoplasm where vigorous streaming occurs, andtheir orientation in the direction of streaming (Ledbetter & Porter, 1963, 1964). It hasbeen suggested that they serve a cytoskeletal function, providing a framework alongwhich the motive force for streaming may be generated (Cronshaw, 1965 a). Thisframework 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 befunctional in streaming and may arise from one another in the cell.

There has been no report dealing with the ultrastructure of coenocytic algae. Thecomplex morphological development of Caulerpa makes it an ideal organism for suchstudies. 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 andregeneration has also been subjected to some investigation and speculation (Dostal,19296; Janse, 1904). This report is concerned with observed characteristics ofstreaming in this organism and a possible association of microtubules with thisphenomenon. For a morphological description of C. prolifera the reader is referred toFritsch (1935), Dostal (1945) or Jacobs (1964).

MATERIAL AND METHODS

C. prolifera (Forsskal) Lamouroux was obtained from the coastal waters of KeyLargo, Florida, and cultured in this laboratory. The algae were grown in synthetic seawater 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 algaewere cleaned and transferred to fresh medium.

It was practicable to follow cytoplasmic streaming only in the leaf of the alga, asthis structure is flat enough to be examined under the microscope and the cell wall inthis region is thinner and much less opaque than in the rhizome. It is assumed that thegeneral pattern and rate of streaming is similar in other regions of the cell. The leafwas isolated by forming a ' pressure wall' (Jacobs, 1964) at the junction of the leaf andrhizome, and excising the former. Streaming was followed and timed over variousregions of the leaf by tracing the path of starch grains or chloroplasts under themicroscope with the aid of a micrometer eyepiece and a stop-watch.

For electron microscopy, segments about 2 cm in length were isolated by pressurewalls from various regions of the rhizome and the cylindrical petiolar region of theleaf. These segments were excised and fixed for 5 min in a refrigerated solution of6-5 % 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 eachtissue segment, smaller pieces (about 1-5 mm long) were cut and returned to theglutaraldehyde fixative for 2-3 h at 3 °C. Small slivers from the leaf lamina were cutdirectly into the fixative. The tissue was washed for 3 h in o-1 M cacodylate buffercontaining 0-25 M sucrose. Secondary fixation was in cold 1% osmium tetroxidesimilarly buffered. The tissue was dehydrated in ethanol or acetone and embedded inEpon 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 in50 % ethanol (20 min) followed by Reynold's lead citrate (20 min). Grids wereexamined in a Hitachi HS-7S electron microscope operating at an acceleratingvoltage of 50 kV.

OBSERVATIONS

Cytoplasmic streaming

The leaf of C. prolifera is characterized by a cylindrical petiolar region at the basethat expands into a flat lamina. At the apex, the leaf often has a depression or notchthat 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 variesbetween 10 and 15 /i in thickness, and below it the parietal cytoplasm consists of astationary ectoplasmic layer, about 5-10 [i in depth, and an endoplasmic layer withinwhich numerous two-way streams are oriented in two distinct patterns. Ovoidchloroplasts, 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 ortrabeculae, possibly with a skeletal function. The cytoplasm also extends over thesurface of the trabeculae and encloses a large central vacuole that extends throughoutthe cell. Electron micrographs indicate that the tonoplast is extremely convoluted inoutline, allowing tenuous fingers of the vacuole to penetrate into the cytoplasmic layers.

In the leaf several distinct longitudinally running streams are visible (Fig. 1). Thesestreams may be as much as 100 /i wide, and the broader streams in the mid-axis mayeach contain 20 moving files of chloroplasts across their width. Where the blade iswidest, the outer longitudinal streams tend to diverge towards the leaf edge, forminga 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, theangle 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 starchgrains. Owing to the thickness and opacity of the material, the migration of organellescould be followed across only one surface of the leaf. However, careful examination ofboth leaf surfaces suggest that the 'slanted streams' actually trace a helical course. Atleast this form of streaming in Caulerpa is distinctive in contrast to the rotationalcyclosis described in Nitella (Kamiya, 1959; Nagai & Rebhun, 1966) and many otheralgae. Towards the edges of the leaves some branching and fusion of streams isapparent.

The longitudinal streams lie at a level below the spiral streams within the interiorof 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 phenomenonis a curious one and calls for closer examination. At this stage, any speculation as tothe morphogenetic function of this asymmetry would be pointless.

The direction of movement in the longitudinal streams seems fairly clear as thestreams 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 thebase-to-apex direction, whereas the lower ones move in the reverse direction. Thespiral streams are also located in at least two different adjacent levels within theendoplasm and movement is bidirectional. However, the narrow streams run so closetogether in both the horizontal and vertical axes that it is difficult to decide whetherthe pattern in this case also is one of separate cytoplasmic layers streaming in oppositedirections.

The rate of streaming in Caulerpa is relatively slow, varying from 3 to 5 /i/sec, ascompared with 60 /i/sec in Nitella (Kamiya, 1959). As a general rule, it appears to bemore rapid in younger, growing leaves that it is in mature leaves.

30-2


Cytoplasmic microtubules

Various techniques of fixation were attempted and the methods finally employedappear to provide the best general preservation of the cytoplasmic contents. Chloro-plasts, nuclei, mitochondria and the Golgi complex were well preserved. There waslittle vesiculation of the cytoplasm, which abounded in ribosomes, generally aggregatedinto 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 unlessthe tissue is post-osmicated and may possibly contain a lipid component. None of thefixation techniques tested could prevent some detachment of the plasma membranefrom the cell wall, but the former generally remained intact. Detailed observations onthe 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 bundleswas 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. Thearea seen is representative of the leaf endoplasm adjacent to the central vacuole. In thisregion, the cytoplasm is extensively penetrated by the vacuole, and restricted tostrands containing the arrays of microtubules and connecting the scattered organelles.By contrast, in the dense cytoplasm adjoining the cell wall, the organelles are closelypacked. In this cortical region, the chloroplasts tend to be aligned with their long axesperpendicular to the wall. On the other hand, as seen in Fig. 3, the chloroplasts in thevicinity of the bundles of microtubules lie parallel to the latter, a feature invariablyobserved in our preparations. The bundles of tubules may be traced over a distanceof 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 verysimilar to those reported for similar structures in other plant material (Ledbetter& Porter, 1963, 1964; Nagai & Rebhun, 1966). The electron-dense wall of the tubuleencloses a less-dense lumen (Figs. 5, 6). The microtubules also run for considerabledistances with little bending, as has been observed before (Burton, 1966; Cronshaw,1965 a), and which suggests a rigid structure. The microtubules in a bundle areusually separated by a constant space of about 300-400 A. The presence of helicallyarranged 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 butare also found in the petiolar region and throughout the rhizome extending to thegrowing tip (Figs. 5, 6). A striking feature is their occurrence in bundles only in theinternal cytoplasm some distance from the cell wall, and often adjacent to the tonoplast(Figs. 6, 7). We have, however, very occasionally observed microtubules in associationwith and possibly parallel to the plasma membrane (Fig. 9). These structures are notaggregated into bundles and appear to be sparsely distributed in a single layer adjacentto the plasmalemma. As a general observation, the endoplasmic bundles appear to liemore or less parallel to one another (Fig. 8) and run in a direction somewhat oblique


to the long axis of the leaf and rhizome. However, owing to difficulties encounteredduring embedding and sectioning in accurately orienting the fragments of tissueexcised from the cell, we are not yet in a position to relate conclusively the orientationof the microtubules and the cytoplasmic streams seen in Figs. 1 and 2.

Except for the arrays of microtubules, no other differences were noticed in thefine structure of the ectoplasm and the endoplasm. No structures correspondingto the 50-A microfilaments described by Nagai & Rebhun (1966) in Nitella wereobserved.

DISCUSSION

In the discussion that follows we shall consider briefly some cellular functions thathave been attributed to microtubules and attempt to justify the suggestion that theyserve a cytoskeletal function in Caulerpa, with a direct or indirect influence on cyto-plasmic streaming. A belief that microtubules may be associated with streaming doesnot imply accrediting these structures with generating the motive force responsiblefor it. Evidence is now accumulating to suggest that within some cells microtubulesmay possibly provide the structural framework that directs the orientation of morethan one phenomenon. Since the original suggestion of Ledbetter & Porter (1963,1964) that microtubules might exert an influence on the disposition of cell-wallmaterial, a number of publications have pointed out that the orientation of corticalmicrotubules 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 beinvolved 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 theplasmalemma at both ends, undermining the likelihood that they are functional in atransporting role. He suggests instead that the oriented skeleton may trap and directwall metabolities, in addition to being concerned with either the generation of motiveforce or the directing of cytoplasmic streaming. Newcomb & Bonnett (1965) foundthat in the young root hairs of radish, the microfibrils of the inner wall layer and theadjacent microtubules were similarly oriented some distance behind the tip. However,the oriented microtubules also extend into the zone near the tip where the wallstructure consists of random microfibrils.

It may be mentioned here that the cell walls of Caulerpa are extremely atypical inthat 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, theirarrangement is on the whole random. The microfibrils within the trabeculae lie bandedtogether in close arrays parallel to the trabecular axis. However, the outer surface of eachtrabeculum 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 wereinvolved.

47O D. D. Sabnis and W. P. Jacobs

What evidence, then, can we muster that may suggest that microtubules are involvedin cytoplasmic streaming? It seems significant that in Caulerpa whereas the corticalregion contains very few microtubules, the inner portion extending up to the tonoplast,and presumably representing the streaming endoplasm, is filled with prominentbundles of regularly arranged and distinctively oriented microtubules. Save for theseelements, no other ultrastructural features distinguish ectoplasm from endoplasm.

In relation to the proposed cytoskeletal function of microtubules, their rigidity isemphasized by the recent observation in the lung-fluke sperm (Burton, 1966), thatincreased periods of sonic disruption result in shorter and shorter fragments of micro-tubules that break transversely and retain their basic structure. In Caulerpa, thepresence 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 thecell is suggested by the occurrence of homologous structures in relation to pronouncedasymmetries in cell forms (Porter, Ledbetter & Badenhausen, 1964). In their possibleparticipation in the directional migration of cytoplasmic materials, these structuresmay 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 ina coenocytic cell like Caulerpa, where growth along the longitudinal axis is accompaniedby regularly spaced, timed and oriented differentiation of the rhizome, it would notbe surprising to find a microtubular framework of the proportions described here.Some similarities between the bundles of microtubules found in Caulerpa and thesimilar arrays of microtubules described in the developing oocyte stalk of the fresh-water mussel (Beams & Sekhon, 1966) are rather striking. Tilney & Porter (1965) arealso of the view that as cells undergo linear extension, microtubules are often arrangedin the direction of the forming extension. In Caulerpa a pronounced polarity ofdevelopment and regeneration exists. It is difficult to visualize a mechanism responsiblefor the expression of polarity other than the directed migration of cytoplasmic com-ponents. Indeed, studies on regeneration in this organism led earlier authors to proposethis hypothesis more than fifty years ago (Dostdl, 1929a, b; Janse, 1890, 1904).

A last point to relate morphogenesis, microtubules and cytoplasmic streaming is theobservation that surgically-induced diversions in streaming patterns of the leaf effectprofound 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 differentiationand serve to provide either the actual framework or to delimit areas of cytoplasmicsubstrate upon which the motive force responsible for streaming is generated.

This investigation was supported by funds from a contract between the Office of NavalResearch, Department of the Navy, and Princeton University. The helpful suggestions ofDr L. I. Rebhun during the course of this work and preparation of the manuscript are gratefullyacknowledged. We are also grateful for the technical assistance of Mr E. Van Norman and theuse of facilities provided by the Whitehall Foundation.


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 cellwall 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 differentiatingxylem elements. In Cellular Ultrastructure of Woody Plants, pp. 99-124. New York: SyracuseUniversity 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 green algae. 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 Coleuscells 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 ' microtubule' 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.

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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-

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TILNEY, L. G. & PORTER, K. R. (1965). Studies on microtubules in Heliozoa. 1. The finestructure of Actinosphaeriuvi nucleofilum (Barrett) with particular reference to the axial rodstructure. Protoplasma 60, 317-344.

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[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 intothe flat lamina. The spiral streams are clearly discernible, x 10.

D. D. SABNIS AND W. P. JACOBS {Facing p. 472)


Fig. 3. Section through the leaf showing the prominent bundles of microtubules(arrows) in the cytoplasm adjacent to the central vacuole. Note the parallel orientationof the chloroplasts lying adjacent to the bundles, (c, Chloroplast; t, trabeculum;v, vacuole.) x 7500.

D. D. SABNIS AND W. P. JACOBS


Fig. 4. Section through the leaf showing portion of a large bundle of tubules. Thearrow points to a cross-banded appearance or a periodicity in the substructure of themicrotubules. x 48 000.Fig. 5. Transverse section of the rhizome. An endoplasmic strand of cytoplasm isfilled with microtubules. x 48000.



Fig. 6. Transverse section of the rhizome. The tonoplast (t) lining the vacuole (v) ispreserved intact. Numerous microtubules (arrow), sectioned transversely, are seenconcentrated in the cytoplasm bounded by the tonoplast. x 63 000.Fig. 7. Section through the leaf. Microtubules are again visible adjacent to thetonoplast, this time sectioned longitudinally, (r, Chloroplast.) X 48 000.



pm

Fig. 8. Section through the leaf, showing the substructure of the microtubules at ahigher magnification, x 96000.Fig. 9. Leaf section near the cell wall. Peripheral microtubules (mt) are visible in closeassociation with the plasmalemma (pm). x 48000.


cytoplasmic streaming and microtubules in the … · marine alga, caulerpa prolifera d. d. sabnis...

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