y. Cell Set. 74, 219-237 (1985) 219 Printed in Great Britain © Company of Biologists Limited 1985 REGULATION OF CELL SHAPE IN EUGLENA GRACILIS. III. INVOLVEMENT OF STABLE MICROTUBULES CAROLE L. LACHNEY AND THOMAS A. LONERGAN* Department of Biological Sciences, University of New Orleans, Lakefnmt, Netu Orleans, LA 70148, U.SA. SUMMARY The role of cytoplasmic microtubules in a recently reported biological clock-controlled rhythm in cell shape of the alga Euglena gracilis (strain Z) was examined using indirect immunofluorescence microscopy. The resulting fluorescent patterns indicated that, unlike many other cell systems, Euglena cells apparently change from round to long to round cell shape without associated cyto- plasmic microtubule assembly and disassembly. Instead, the different cell shapes were correlated with microtubule patterns, which suggested the movement of stable microtubules to accomplish cell shape changes. In live intact cells, these microtubules were demonstrated by immunofluorescence to be stable to lowered temperature and elevated intracellular Ca2"1" levels, treatments that are commonly used to depolymerize microtubules. In cells extracted in detergent at low temperature or in the presence of elevated Ca2+ levels, the fluorescent image of the microtubules was disrupted. Transmission electron microscopy confirmed the loss of one subset of pellicle microtubules. The difference in microtubule stability to these agents between live intact cells and cells extracted in detergent suggested the presence of a microtubule-stabilizing factor in live cells, which is released from the cell by extraction with detergent, thereby permitting microtubule depolymerization by Ca2+ or lowered temperature. The calmodulin antagonist trifluoperazine prevented the Ca2+- induced disruption of the fluorescent microtubule pattern in cells extracted in detergent. These results implied the involvement of calmodulin in the sensitivity to Ca2+ of the microtubules of cells extracted in detergent. INTRODUCTION The determination of cell shape is thought to involve the specific arrangement of the microtubule component of the cytoskeleton. Changes in cell shape are thought to result from a reorganization of the microtubule cytoskeleton. Many studies have supported the conclusion that an increase in cellular asymmetry (anisometry) is accompanied by microtubule polymerization and a decrease in asymmetry, or cell rounding, is accompanied by microtubule depolymerization (Porter, 1966,1980). These studies typically employ microtubule-disrupting agents to remove the micro- tubule component of the cytoskeleton. Cell shape changes, such as the retraction of heliozoan axopods (Schliwa, 1976) or neuroblastoma neurites (Olmsted, 1981) in response to low temperature, or the rounding of normally pyriform Ochromonas cells (Brown & Bouck, 1973) as a result of exposure to colchicine or increased pressure, •Author for correspondence. Key words: microtubules, cell shape, Euglena. 220 C. L. Lachney and T. A. Lonergan accompany the observed disruption of microtubules. The conclusion that the loss of microtubules is related to the loss of cellular asymmetry is strengthened by the observation that removal of the microtubule-disrupting agents permits the recovery of normal cell morphology as well as repolymerization of the microtubules. Only a few studies have been performed with naturally occurring shape changes, i.e. those not chemically or physically induced. The transition from round to flattened mouse 3T3 fibroblasts will occur if cells are permitted to settle onto a solid substrate. The microtubule cytoskeleton, which closely matches the changing cell shape, is observed to polymerize during the cell spreading event (Osborn & Weber, 1976). Mouse neuroblastoma cells begin to extend neurites when permitted to settle onto a solid substrate, and these neurites contain bundles of polymerizing microtubules (Olmsted, 1981; Solomon &Magendantz, 1981). In the colonial alga Volvox, daughter colonies invert, a process that is characterized by elongation of individual cells in the colony. Within the inverting cells are microtubules that are apparently synthesized as cell elongation takes place and lie parallel to the axis of elongation (Viamontes, Focht- mann & Kirk, 1979). A new system has been reported in which Euglena gracilis cells change shape between two extremes, round and long cells, every 24 h (Lonergan, 1983). The time scale (h) of the cell shape change makes it amenable to ultrastructural study. Colchicine inhibits the cell shape changes inEuglena (Lonergan, 1983), imply- ing the involvement of microtubules, but without microscopic examination of the microtubules this involvement was a matter of speculation. The present study was undertaken to examine the role of microtubules in these shape changes, which are at least superficially similar to the shape changes of metaboly (Guttman & Ziegler, 1974; Hofmann & Bouck, 1976), but slower. This article reports our characterization of the pellicle microtubules of E. gracilis by indirect immunofluorescence. We have found a correlation between cell shape and microtubule arrangement that, unlike the above systems, apparently did not involve the polymerization and depolymerization of microtubules. Examination of microtubule arrangements of different cell shapes suggested instead the movement of a stable population of microtubules by intracellular contractile forces. We further characterized the stability of the pellicle microtubules to commonly used microtubule-disrupting agents. In addition to stability to colchicine, previously repor- ted by Silverman & Hikida (1976), we found Euglena microtubules to be stable to low temperature and elevated intracellular Ca2+ levels in live cells. Exposure of detergent- extracted cells to elevated Ca2+ levels or low temperature resulted in loss of micro- tubules, suggesting the existence of an extraction-labile stabilizing factor. Experiments with the calmodulin antagonist trifluoperazine have implicated calmodulin in the Ca2+ - induced depolymerization of microtubules in Euglena cells extracted in detergent. MATERIALS AND METHODS Cell cultures Axenic cultures of Euglena gracilis (strain Z) were grown from samples of liquid stocks in 250 ml flasks containing 175 ml Cramer—Myers liquid culture medium (Cramer & Myers, 1952). Cultures Stable microtubules in Euglena 221 were aerated with filter-sterilized air at an approximate rate of 0-51 min"1, magnetically stirred, and maintained at 25°C. The illumination consisted of a 10-h light/14-h dark cycle at fluorescent light intensity of 300 /iEinsteins m~2 s"1. Fluorescent antibody staining Samples for antibody staining consisted of 20 ml portions at a density of 0-75 X 10s to 1-0 X 105 cells/ml (late log phase), removed at the times of the extremes of the cell shape rhythm, 0900 standard time (ST, 9:00am, round cells, corresponding to lights-on of the illumination cycle), 1400 ST (2:00 pm, long cells, 5 h into the light period) or at times between the extremes of shape. In order to prevent cell shape changes, control cells or cells exposed to various experimental conditions were fixed in suspension at room temperature by slowly adding equal volumes of 6% (w/v) paraformaldehyde to the cell suspension while gently mixing on a vortex mixer, resulting in a final 3% (w/v) paraformaldehyde solution. Cells were collected by centrifugation in a Damon IEC clinical centrifuge at maximum setting for 30 s and gently resuspended in a second fixative consisting of 3% (w/v) paraformaldehyde in a microtubule-stabilizing buffer consisting of lOOmM-PIPES (pH 6-9), 1 mM-MgS04, 2mM-EGTA, 330 mM-sorbitol (Lloyd etal. 1979; Lloyd, Slabas, Powell & Lowe, 1980), for 10min. Cells were centrifuged and resuspended in microtubule-stabilizing buffer without sorbitol, containing 2-5% Triton X-100 (v/v), and extracted for 15min while agitating in a gyratory shaker (175 rev./min). All subsequent centrifugations were at 1600 rev./min (500 g), for 10 min in a Beckman TJ-6 tabletop centrifuge. After extraction, cells were centrifuged and resuspended in 5 ml 80% (v/v) acetone for 5 min while agitating in a gyratory shaker to remove chlorophyll, which causes an interfering red fluorescence. Cells were then centrifuged and re- suspended twice for 5 min, while agitating in 5 ml microtubule buffer without sorbitol (to hasten extraction of soluble cytoplasmic contents), followed by three additional resuspensions (for 5 min) in microtubule buffer containing 0-lmM-glycine (pH 7-4) to reduce excess aldehydes. Cell pellets were then rinsed twice in microtubule buffer and centrifuged in a Beem capsule to obtain a 1 - 2/il pellet. These cells were incubated in 50 fA sheep anti-bovine brain tubulin (Caabco, Inc., Houston, TX, 225^g/ml antibody in 125 mM-borate and 75mM-NaCl, pH 8-4) for 45 min at 25°C. Cells were rinsed three times in 10 drops of microtubule buffer and resuspended in 50fil FITC- conjugated rabbit anti-sheep IgG (Miles-Yeda, Rehovot, Israel, 100-200 ^g/ml specific antibody in lOmM-phosphate and 75mM-NaCl, pH 7-4) 45 min at 25°C. Cells were rinsed three times in 10 drops of microtubule buffer, and resuspended for storage in 1 drop of microtubule buffer containing 1% (w/v) NaN3. Cells were stored refrigerated in covered Beem capsules. Cells were viewed by making wet mounts of approximately 1 /A cell suspension and 1 (A mounting medium consisting of 1% (w/v) />-phenylenediamine in glycerol at pH 8. Cells were viewed and photographed with an Olympus Model BHT microscope with