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Cytoplasmic Streaming and Microtubules in the Coenocytic Marine Alga, Caulerpa Prolifera

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Cytoplasmic Streaming and Microtubules in the Coenocytic Marine Alga, Caulerpa Prolifera J. Cell Sci. 2, 465-472 (1967) 465 Printed in Great Britain 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 through- out 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 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 microfilaments in the plant cell 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 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 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.
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