MICROTUBULE BIOGENESIS AND CELL SHAPE IN OCHROMONAS

Abstract

A B S T R A C TThe proposal made in the preceding paper that the species-specific shape of Ochromonas is mediated by cytoplasmic microtubules which are related to two nucleating sites has been experimentally verified. Exposure of cells to colchicine or hydrostatic pressure causes microtubule disassembly and a correlative loss of cell shape in a posterior to anterior direction. Upon removal of colchicine or release of pressure, cell shape regenerates and microtubules reappear, first in association with the kineto-beak site concomitant with regeneration of the anterior asymmetry, and later at the rhizoplast site concomitant with formation of the posterior tail. It is concluded that two separate sets of cytoplasmic tubules function in formation and maintenance of specific portions of the total cell shape. On the basis of the following observations, we further suggest that the beak and rhizoplast sites could exert control over the position and timing of the appearance, the orientation, and the pattern of microtubule distribution in Ochromonas. (a) the two sites are accurately positioned in the cell relative to other cell organelles; (b) in regenerating cells microtubules reform first at these sites and appear to elongate to the cell posterior; (c) microtubules initially reappear in the orientation characteristic of the fully differentiated cell; (d) the two sets of tubules are polymerized at different times, in the same sequence, during reassembly or resynthesis of the microtubular system. Experiments using cycloheximide, after a treatment with colchicine, have demonstrated that Ochromonas cannot reassume its normal shape without new protein synthesis. This suggests that microtubule protein once exposed to colchicine cannot be reassembled into microtubules. Pressure-treated cells, on the other hand, reassemble tubules and regenerate the normal shape in the presence or absence of cycloheximide. The use of these two agents in analyzing nucleating site function and the independent processes of synthesis and assembly of microtubules is discussed.The preceding paper (Bouck and Brown, 1973) examines the fine structure of vegetative and dividing cells of Ochromonas and describes, (a) a close correlation between the distribution of cytoplasmic microtubules and the asymmetric cell shape, and (b) the association of these microtubules with two distinct sites in the cell anterior. These observations implied a cytoskeletal role for microtubules and further suggested that control of microtubule distribution, and hence control of cell shape, may be effected by restricting microtubule assembly to specific nucleating sites (terminology of Tilney and Goddard, 1970).The present study was undertaken to determine if the observed microtubule distribution is obligatory to development and/or maintenance of cell shape, as well as to clarify whether the presumptive nucleating sites do in fact function as sites of initial microtubule assembly. It was found that microtubule depolymerizing agents (colchicine 860

The Energetics of Freshwater Algae; Energy Requirements for Biosynthesis and Volume Regulation

“…With an external osmolarity of 2 osmol m^^ (cf. Bowen, 1979) the driving force for water entrj' is 0-2 MPa. With an Lp value of 10^" m s^i Pa'^ (cf.…”

“…Functional cytoplasm has an osmolarity in excess of some 40 osmol m~^ (Prosser, 1973;Raven, 1974Raven, , 1976Raven, , 1980, while 'freshwater' has an osmolarity of 2-0 to 2-5 osmol m"^ (based on the composition of 'average' freshwater: Bowen, 1979). According, living cells in a freshwater medium are hypertonic to their medium, with a corresponding tendancy for water to enter the cell (osmosis).…”

Microtubular Organization in Elongating Myogenic Cells

“…An attempt to quantify the extent of paraxial association between microtubules at different levels in the same cell and between different myogenic cells has been made by counting numbers of microtubules lying within about 40 nm (surface-tosurface) of one another in transverse sections. The ratios of paraxially associated microtubules to total numbers of microtubules in consecutive transverse sections have been plotted as a function of cell length for five cells (4,5,6,7,8) (Fig. 9).…”

“…play in supporting cell shape, particularly with regard to cell elongation (4,7,12,8), and the formation and maintenance of long processes from the cell surface (36,41), and it has been shown with the use of antimicrotubule agents that disruption of microtubules in anisometric cells results in reversible cell shortening or withdrawal of cell processes (5,20,37,38,40). In the systems studied, the common feature of microtubule organization is an orientation of microtubules in the axis of the cellular anisometry, although there may be varying degrees of more complex interactions among the aligned microtubules.…”

Regulation of Cell Shape in Euglena gracilis

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