Temperature-Sensitive Chlamydomonas Mutants Manifesting Flagellar Regression at a Restrictive Temperature‡

The regeneration kinetics of Chlamydomonas reinhardtii mutants TS-6 and TS-79, whose flagella were mechanically amputated, indicated that the flagellar precursor in cytoplasm was used for regeneration when cycloheximide was present. The TS-6 cells rendered nonflagellate by regression at 35 C did not regenerate in the presence of cycloheximide, indicating that the precursor was inactivated by the high temperature. Neither mutant was able to use the absorbed flagellar components for regeneration in the presence of cycloheximide.     

Hydrostatic pressure-induced internalization of flagellar axonemes, disassembly, and reutilization during flagellar regeneration in Polytomella

Exposure of the quadriflagellate Polytomella to hydrostatic pressure was shown to result in the internalization of intact flagellar axonemes. During recovery from the pressure treatment the axonemes were disassembled concurrent with flagellar regeneration. When flagella were amputated partial regeneration occurred in the presence of cycloheximide, suggesting the presence of a limiting available pools of flagellar precursors. After a second amputation in the continued presence of cycloheximide little or no regeneration occurred, indicating depletion of the pool. However, if internalized axonemes were available, as well as the precursor pool, full-length flagella regenerated in cycloheximide. When the pool had been depleted and internalized axonemes were present, flagella regenerated to a length equal to the initial length of the internalized axonemes. We conclude that materials resulting from the disassembly of the pressure internalized axonemes are reutilized in regenerating new flagella.     

FLAGELLAR REGENERATION IN PROTOZOAN FLAGELLATES

The flagella of populations of three protozoan species (Ochromonas, Euglena, and Astasia) were amputated and allowed to regenerate. The kinetics of regeneration in all species were characterized by a lag phase during which there was no apparent flagellar elongation; this phase was followed by elongation at a rate which constantly decelerated as the original length was regained. Inhibition by cycloheximide applied at the time of flagellar amputation showed that flagellar regeneration was dependent upon de novo protein synthesis. This was supported by evidence showing that a greater amount of leucine was incorporated into the proteins of regenerating than nonregenerating flagella. The degree of inhibition of flagellar elongation observed with cycloheximide depended on how soon after flagellar amputation it was applied: when applied to cells immediately following amputation, elongation was almost completely inhibited, but its application at various times thereafter permitted considerable elongation to occur prior to complete inhibition of flagellar elongation. Hence, a sufficient number of precursors were synthesized and accumulated prior to addition of cycloheximide so that their assembly (elongation) could occur for a time under conditions in which protein synthesis had been inhibited. Evidence that the site of this assembly may be at the tip of the elongating flagellum was obtained from radioautographic studies in which the flagella of Ochromonas were permitted to regenerate part way in the absence of labeled leucine and to complete their regeneration in the presence of the isotope. Possible mechanisms which may be operating to control flagellar regeneration are discussed in light of these and other observations.     

FLAGELLAR ELONGATION AND SHORTENING IN CHLAMYDOMONAS : The Use of Cycloheximide and Colchicine to Study the Synthesis and Assembly of Flagellar Proteins

Flagella can be removed from the biflagellate Chlamydomonas and the cells begin to regenerate flagella almost immediately by deceleratory kinetics. Under usual conditions of deflagellation, more than 98% of all flagella are removed. Under less drastic conditions, cells can be selected in which one flagellum is removed and the other left intact. When only one of the two flagella is amputated, the intact flagellum shortens by linear kinetics while the amputated one regenerates. The two flagella attain an equal intermediate length and then approach their initial length at the same rate. A concentration of cycloheximide which inhibits protein synthesis permits less than one-third of each flagellum to form when both flagella are amputated. When only one is amputated in cycloheximide, shortening proceeds normally and the degree of elongation in the amputated flagellum is greater than if both were amputated in the presence of cycloheximide. The shortening process is therefore independent of protein synthesis, and the protein from the shortening flagellum probably enters the pool of precursors available for flagellar formation. Partial regeneration of flagella occurs in concentrations of cycloheximide inhibitory to protein synthesis suggesting that some flagellar precursors are present. Cycloheximide and flagellar pulse-labeling studies indicate that precursor is used during the first part of elongation, is resynthesized at mid-elongation, and approaches its original level as the flagella reach their initial length. Colchicine completely blocks regeneration without affecting protein synthesis, and extended exposure of deflagellated cells to colchicine increases the amount of flagellar growth upon transfer to cycloheximide. When colchicine is applied to cells with only one flagellum removed, shortening continues normally but regeneration is blocked. Therefore, colchicine can be used to separate the processes of shortening and elongation. Radioautographic studies of the growth zone of Chlamydomonas flagella corroborate previous findings that assembly is occurring at the distal end (tip growth) of the organelle.     

Flagellar regeneration in Chlamydomonas reinhardtii: evidence that cycloheximide pulses induce a delay in morphogenesis.

The behaviour of a pool of flagellar precursors, assayed by the ability of cells to regenerate flagella in the absence of de novo protein synthesis, has been examined during organelle morphogenesis in the biflagellate alga Chlamydomonas. The results demonstrate that flagellar elongation can continue even when this pool is apparently empty and suggest that 2 sources of precursors are available to the regenerating flagella: those pre-existing in the cellular pool and those synthesized de novo. Further evidence for this was obtained by subjecting regenerating cells to pulses of cycloheximide. Cells exposed to this drug during the first 60 min post deflagellation formed only half-length (5-mum) flagella, whereas a pulse administered after this point allowed the formation of longer flagella and suggested that some de novo protein synthesis was required for the formation of full-length flagella, although it was not a prerequisite for the initiation of regeneration. In addition, it was found that, subsequent to the removal of the cycloheximide, flagellar regeneration did not recommence immediately, but was delayed for a period of approximately 45 min, irrespective of length of flagella formed prior to drug inhibition. The nature of this cycloheximide-induced delay is unclear and certain alternatives, based on the exhaustion of structural/regulatory components are considered. Although it is not possible to distinguish between these alternatives, tubulin is not the limiting component, since a pool of this protein is present when flagellar elongation is prevented by cycloheximide.     

Flagellar root contraction and nuclear movement during flagellar regeneration in Chlamydomonas reinhardtii

When Chlamydomonas cells are deflagellated by pH shock or mechanical shear the nucleus rapidly moves toward the flagellar basal apparatus at the anterior end of the cell. During flagellar regeneration the nucleus returns to a more central position within the cell. The nucleus is connected to the flagellar apparatus by a system of fibers, the flagellar roots (rhizoplasts), which undergo a dramatic contraction that coincides with anterior nuclear movement. A corresponding extension of the root system, back to its preshock configuration is observed as the nucleus retracts to a central position. Anterior displacement of the nucleus and flagellar root contraction require free calcium in the medium. Nuclear movement and flagellar root contraction and extension are not sensitive to inhibitors of protein synthesis (cycloheximide), or drugs that influence either microtubules (colchicine) or actin-based microfilaments (cytochalasin D). Detergent-extracted cell models contract and extend their flagellar roots and move their nuclei in response to alterations of free calcium levels in the medium. Cycles of nuclear movement in detergent-extracted models require ATP to potentiate the contractile mechanism for subsequent calcium-induced contraction. Flagellar root contraction and nuclear movement in Chlamydomonas may be causally related to signaling of induction of flagellar precursor genes or to the transport of flagellar precursors or their messages to sites of synthesis or assembly near the basal apparatus of the cell.     

Flagellar regeneration in the scaly green flagellateTetraselmis striata (Prasinophyceae): regeneration kinetics and effect of inhibitors

Flagellar regeneration after experimental amputation was studied in synchronized axenic cultures of the scaly green flagellateTetraselmis striata (Prasinophyceae). After removal of flagella by mechanical shearing, 95% of the cells regrow all four flagella (incl. the scaly covering) to nearly full length with a linear velocity of 50 nm/min under standard conditions. Flagellar regeneration is independent of photosynthesis (no effect of DCMU; the same regeneration rate in the light or in the dark), but depends on de novo protein synthesis: cycloheximide at a low concentration (0.35 μM) blocks flagellar regeneration reversibly. No pool of flagellar precursors appears to be present throughout the flagellated phase of the cell cycle. A transient pool of flagellar precursors, sufficient to generate 2.5 μm of flagellar length, however, develops during flagellar regeneration. Tunicamycin (2 μg/ml) inhibits flagellar regeneration only after a second flagellar amputation, when flagella reach only one third the length of the control. Flagellar regeneration inT. striata differs considerably from that ofChlamydomonas reinhardtii and represents an excellent model system for the study of synchronous Golgi apparatus (GA) activation, and transport and exocytosis of GA-derived macromolecules (scales).     

On the localization of voltage-sensitive calcium channels in the flagella of Chlamydomonas reinhardtii

This study was undertaken to prove that voltage-sensitive calcium channels controlling the photophobic stop response of the unicellular green alga Chlamydomonas reinhardtii are exclusively found in the flagellar region of the cell and to answer the question as to their exact localization within the flagellar membrane. The strategy used was to amputate flagella to a variable degree without perturbing the electrical properties of the cell and measure flagellar currents shortly after amputation and during the subsequent regeneration process. Under all conditions, a close correlation was found between current size and flagellar length, strongly suggesting that the channels that mediate increases in intraflagellar calcium concentration are confined to and distributed over the total flagellar length. Bald mutants yielded tiny flagellar currents, in agreement with the existence of residual flagellar stubs. In the presence of the protein synthesis inhibitor cycloheximide, flagellar length and flagellar currents also recovered in parallel. Recovery came to an earlier end, however, leveling off at a time when in the absence of cycloheximide only half maximal values were achieved. This suggests the existence of a pool of precursors, which permits the maintenance of a constant ratio between voltage-sensitive calcium channels and other intraflagellar proteins.     

A conditional cell division mutant of Chlamydomonas reinhardii having an increased level of colchicine resistance

Conditional cell division mutants were isolated from Chlamydomonas reinhardii. They were unable to form colonies at 34 °C but not at 23 °C. One of the mutants, TS-60, could neither divide at high nor at low (15 °C) temperature, and seemed to continue protein synthesis at restrictive temperatures. TS-60 also exhibited resistance to 6 mM colchicine which inhibited cell division of the wild-type. Observing that TS-60 flagella were highly resistant to colchicine in their regeneration, it is concluded that the mutational alteration has affected not only the mitotic apparatus but also the flagella. Thermolability of TS-60 was not detected in flagellation but in cell division, though colchicine resistance was expressed in both flagellation and cell division. This suggests that the stable formation of the flagellar microtubule mainly depends on the specific organization of its component. Both thermolability and colchicine resistance of TS-60 were inherited in a Mendelian fashion and unseparable from each other. Reversion tests indicated that the two characters were caused by a single mutation. It is inferred that the above-mentioned phenotypes of TS-60 are the consequence of a mutation in factor(s) involving the colchicine binding activity of tubulin and that this mutational change pleiotrophically leads to some impediment in microtubule formation at restrictive temperature.     

The Golgi apparatus of the scaly green flagellate Scherffelia dubia: uncoupling of glycoprotein and polysaccharide synthesis during flagellar regeneration

The flagella of the green alga Scherffelia dubia are covered by scales which consist of acidic polysaccharides and glycoproteins. Experimental deflagellation results in the regeneration of flagella complete with scales. During flagellar regeneration, scales are newly synthesized in the Golgi apparatus, exocytosed and deposited on the growing flagella. Flagellar regeneration is dependent upon protein synthesis and N-glycosylation, as it is blocked by cycloheximide and partially inhibited by tunicamycin. Metabolic labeling with [³⁵S]methionine/cysteine demonstrated that scale-associated proteins were not newly synthesized during flagellar regeneration, suggesting that the proteins deposited on regenerating flagella were drawn from a pool. Quantitative immunoelectron microscopy using a monospecific antibody directed against a scale-associated protein of 126 kDa (SAP126) revealed that the pool of SAP126 was primarily located at the plasma membrane, with minor labeling of the scale reticulum and trans-Golgi cisternae, both before deflagellation and during flagellar regeneration. Since SAP126 was sequestered during flagellar regeneration into secretory vesicles together with newly synthesized scales, it is concluded that the persistent presence of SAP126 in the trans-Golgi cisternae during scale biogenesis requires retrograde transport of the protein from the plasma membrane to the Golgi apparatus. Received: 3 July 1999 / Accepted: 21 August 1999     

How is the flagellar length of mature sperm determined? III. Comparison of initial growth rate of flagella in spermatids from Cynops and Xenopus in the presence and absence of cycloheximide.

Previous studies on flagellar growth in round spermatids from Cynops and Xenopus in vitro have shown that the period and rate of flagellar growth are greater in Cynops than in Xenopus. The present study shows, however, that during the initial phase of flagellar growth (for the first 12 h following the second meiotic division), the growth rate is very similar in both Cynops and Xenopus (0.5-0.6 microns/h at 22 degrees C). The difference in the growth rate between Cynops and Xenopus was observed beyond 12 h following the second meiotic division. When round spermatids in both species were inoculated with 10 microM cycloheximide, flagella grew at the same rate as in the absence of cycloheximide for the first 12 h following the second meiotic division. Beyond 12 h, however, cycloheximide suppressed flagellar growth in round spermatids in both species. These results indicate that the initial flagellar growth in round spermatids is provided for by flagellar protein pools which were present just after the second meiotic division; the growth beyond 12 h in round spermatids is contributed by newly synthesized flagellar proteins.     

Acetylcholine receptors in the central nervous system of Drosophila melanogaster.

Mating between gametes of the biflagellated unicellular green alga Chlamydomonas reinhardi consists of several events culminating in zygote formation. Initially, the cells agglutinate by their flagellar tips. This is followed by pairing, cell wall loss, and cell fusion. Here we report on the relationship between the length of the flagellum, and the cells' ability to agglutinate, undergo cell wall loss (as measured by medium carbohydrate accumulation), and to form zygotes. We found that deflagellated gametes regained the potential for sexual agglutination when the flagella had regenerated to less than 3 μm (compared to the full length flagella of approx. 11 μm), while medium carbohydrate appeared only after the flagella had reached an average length greater than 5 μm. By inhibiting flagellar regeneration with cycloheximide or colchicine, we determined that carbohydrate release is related to the length of the flagellum and not to the time after deflagellation. A flagellar length dependence similar to that of carbohydrate release was also observed when we measured the relationship between the gametes' ability to fuse and flagellar length.     

Flagellar Development During the Cell Cycle in Chlamydomonas reinhardtii

Flagellar development during the asexual synchronous cell cycle of Chlamydomonas reinhardtii (11.32 aM) was studied by light microscopy. Cell walls of sporangia of different developmental status were dissolved using gamete lysin (g-lysin) enabling direct observation of flagellar development. Flagellar growth in progeny cells exhibits a linear kinetic with a growth rate of 28 nm/min at 30°C leading to a flagellar length of 7–7.5 μm in 4–4.5 h. After this time the flagellar growth rate drops to 2.8 nm/min (as in interphase). Both flagella of a single cell and all flagella within a sporangium grow out at the same time and with the same rate. Cycloheximide (10 μg/ml) completely blocks flagellar development. If cycloheximide is removed flagellar growth resumes at the normal rate with no lag-phase. Flagellar development during the cell cycle in C. reinhardtii differs considerably from the well-studied model system of flagellar regeneration following amputation in the same species.     

The influence of the herbicide trifluralin on flagellar regeneration in Chlamydomonas

The mode of action of trifluralin is known to include disruption of cell division in root meristems by causing an absence of spindle microtubules. It has also been shown that trifluralin binds to tubulin isolated and purified from Chlamydomonas flagella. In this paper the kinetics of in vivo flagellar regeneration was used as a model to determine the influence of trifluralin on tubulin assembly. Chlamydomonas cells were grown in synchronous culture using a 12 h light-dark cycle. At 3 h into the light cycle the cells were subjected to shear force to induce flagellar abortion. Flagellar regeneration, in the presence of varying concentrations of trifluralin, was observed by Nomarski interference microscopy. After 1.5 h, trifluralin concentrations below 0.1 μM had not affected the regeneration rate, while concentrations above 5 μM prevented the onset of regeneration. As the concentration between 0.1 and 5 μM was increased, the final length of all flagella decreased. Using combinations of cycloheximide and trifluralin it was determined that trifluralin did not influence tubulin synthesis, and removing trifluralin only restored 50% of the regeneration capacity present at the beginning of treatment. By comparing groups of cells where the tubulin pool was depleted or present, it was found that trifluralin prevented assembly rather than causing a breakdown of previously assembled flagella. The research reported here supports the theory that the mechanism of action of trifluralin is an interaction of trifluralin and tubulin in a way that prevents tubulin assembly into spindle microtubules.     

How is the flagellar length of mature sperm determined? I. Comparison of flagellar growth in spermatids between newt and Xenopus in vitro

The kinetics of flagellar growth in round spermatids were compared between Xenopus laevis and Cynops pyrrhogaster in vitro, the latter of which has about 13 times longer flagella in mature sperm than the former. In both species, more than 90% of the spermatids derived from marked primary spermatocytes grew flagella. In Xenopus the average flagellar length increased to 28 microns by the 6th day and then stopped growth, while in the newt, flagellar growth did not stop until reaching 107 microns in average on the 10th day. Maximal length was 36-38 microns in Xenopus and 187 microns in the newt. Two major differences in kinetics of flagellar growth were found between the two species. First, the initial rate of growth in the newt was about double the rate in Xenopus. Second, the period of flagellar growth in the newt (10 days) was also about double the period in Xenopus (5-6 days). Actinomycin D (10 micrograms/ml) had no inhibitory effect on flagellar growth in either species, whereas cycloheximide (10 microM) inhibited flagellar growth by more than 80% in both species. These results indicate that translational control presumably of flagellar protein synthesis plays an important role in flagellar growth in both species and in the difference in flagellar length in spermatids between Xenopus and newt.     

ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE IN CHLAMYDOMONAS REINHARDTII : Influence on Flagellar Function and Regeneration

Adenosine 3',5'-cyclic monophosphate (cAMP) influences both flagellar function and flagellar regeneration in Chlamydomonas reinhardtii. The methylxanthine, aminophylline, which can cause a tenfold increase in cAMP level in C. reinhardtii, inhibits flagellar movement and flagellar regeneration by wild-type cells, without inhibiting cell multiplication. Caffeine, a closely related inhibitor, also inhibits flagellar movement and regeneration, but it inhibits cell multiplication too. Regeneration by a mutant lacking the central pair of flagellar microtubules was found to be more sensitive than wild type to inhibition by caffeine and to be subject to synergistic inhibition by aminophylline plus dibutyryl cAMP. Regeneration by three out of seven mutants with different flagellar abnormalities was more sensitive than wild type to these inhibitors. We interpret these results to mean that cAMP affects a component of the flagellum directly or indirectly, and that the responsiveness of that component to cAMP is enhanced by mutations which affect the integrity of the flagellum. The component in question could be microtubule protein.     

A Role for the Membrane in Regulating Chlamydomonas Flagellar Length

Flagellar assembly requires coordination between the assembly of axonemal proteins and the assembly of the flagellar membrane and membrane proteins. Fully grown steady-state Chlamydomonas flagella release flagellar vesicles from their tips and failure to resupply membrane should affect flagellar length. To study vesicle release, plasma and flagellar membrane surface proteins were vectorially pulse-labeled and flagella and vesicles were analyzed for biotinylated proteins. Based on the quantity of biotinylated proteins in purified vesicles, steady-state flagella appeared to shed a minimum of 16% of their surface membrane per hour, equivalent to a complete flagellar membrane being released every 6 hrs or less. Brefeldin-A destroyed Chlamydomonas Golgi, inhibited the secretory pathway, inhibited flagellar regeneration, and induced full-length flagella to disassemble within 6 hrs, consistent with flagellar disassembly being induced by a failure to resupply membrane. In contrast to membrane lipids, a pool of biotinylatable membrane proteins was identified that was sufficient to resupply flagella as they released vesicles for 6 hrs in the absence of protein synthesis and to support one and nearly two regenerations of flagella following amputation. These studies reveal the importance of the secretory pathway to assemble and maintain full-length flagella.