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1.
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  相似文献   

2.
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.  相似文献   

3.
Synthesis of new proteins is required to regenerate full length Chlamydomonas flagella after deflagellation. Using gametes, which have a low basal level of protein synthesis, it has been possible to label and detect the synthesis of many flagellar proteins in whole cells. The deflagellation-induced synthesis of the tubulins, dyneins, the flagellar membrane protein, and at least 20 other proteins which co- migrate with proteins in isolated axonemes, can be detected in gamete cytoplasm, and the times of initiation and termination of synthesis for each of the proteins can be studied. The nature of the signal that stimulates the cell to initiate flagellar protein synthesis is unknown. Flagellar regeneration and accompanying pool depletion are not necessary for either the onset or termination of flagellar protein synthesis, because colchicine, which blocks flagellar regeneration, does not change the pattern of proteins synthesized in the cytoplasm after deflagellation or the timing of their synthesis. Moreover, flagellar protein synthesis is stimulated after cells are chemically induced to resorb their flagella, indicating that the act of deflagellation itself is not necessary to stimulate synthesis. Methods were defined for inducing the cells to resorb their flagella by removing Ca++ from the medium and raising the concentration of K+ or Na+. The resorption was reversible and the flagellar components that were resorbed could be re-utilized to assemble flagella in the absence of protein synthesis. This new technique is used in this report to study the control of synthesis and assembly of flagella.  相似文献   

4.
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).  相似文献   

5.
A new ‘paralyzed’ mutant. OC–10, was isolated in Chlamydomonas reinhardtii Dangeard. OC-10 cannot swim and generally shows very little flagellar movement. However, when OC-10 was demembranated, axonemal motility was reactivated in the presence of adenosine triphosphate (ATP) or adenosine diphosphate (ADP). The beating form of the reactivated axonemes was almost the same as that of the wild-type axonemes. Flagellar regeneration of OC-10 was slower than that of the wild-type. Electron microscopic examination showed no abnormality in OC-10 flagella, but SDS/PAGE revealed that mobility of a flagellar membrane protein was changed and a few bands disappeared in OC-10 flagella, When the mutant was crossed to wild-type to form temporary dikaryon cells with 4 flagella, OC-10 flagella did not regain motility. Tetrad analysis of crosses between OC–10 and wild-type demonstrated a 1:1 segregation on the basis of flagellar motility. From these results, we suppose that OC-10 may be limited in ATP availability inside the flagella, or altered in flagellar membrane proteins important for motility.  相似文献   

6.
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.  相似文献   

7.
Polarity of flagellar assembly in Chlamydomonas.   总被引:1,自引:0,他引:1       下载免费PDF全文
During mating of the alga Chlamydomonas, two biflagellate cells fuse to form a single quadriflagellate cell that contains two nuclei and a common cytoplasm. We have used this cell fusion during mating to transfer unassembled flagellar components from the cytoplasm of one Chlamydomonas cell into that of another in order to study in vivo the polarity of flagellar assembly. In the first series of experiments, sites of tubulin addition onto elongating flagellar axonemes were determined. Donor cells that had two full-length flagella and were expressing an epitope-tagged alpha-tubulin construct were mated (fused) with recipient cells that had two half-length flagella. Outgrowth of the shorter pair of flagella followed, using a common pool of precursors that now included epitope-tagged tubulin, resulting in quadriflagellates with four full-length flagella. Immunofluorescence and immunoelectron microscopy using an antiepitope antibody showed that both the outer doublet and central pair microtubules of the recipient cells' flagellar axonemes elongate solely by addition of new subunits at their distal ends. In a separate series of experiments, the polarity of assembly of a class of axonemal microtubule-associated structures, the radial spokes, was determined. Wild-type donor cells that had two full-length, motile flagella were mated with paralyzed recipient cells that had two full-length, radial spokeless flagella. Within 90 min after cell fusion, the previously paralyzed flagella became motile. Immunofluorescence microscopy using specific antiradial spoke protein antisera showed that radial spoke proteins appeared first at the tips of spokeless axonemes and gradually assembled toward the bases. Together, these results suggest that both tubulin and radial spoke proteins are transported to the tip of the flagellum before their assembly into flagellar structure.  相似文献   

8.
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.  相似文献   

9.
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.  相似文献   

10.
11.
Chlamydomonas reinhardtii controls flagellar assembly such that flagella are of an equal and predetermined length. Previous studies demonstrated that lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), induced flagellar elongation, suggesting that a lithium-sensitive signal transduction pathway regulated flagellar length (S. Nakamura, H. Takino, and M. K. Kojima, Cell Struct. Funct. 12:369-374, 1987). Here, we demonstrate that lithium treatment depletes the pool of flagellar proteins from the cell body and that the heterotrimeric kinesin Fla10p accumulates in flagella. We identify GSK3 in Chlamydomonas and demonstrate that its kinase activity is inhibited by lithium in vitro. The tyrosine-phosphorylated, active form of GSK3 was enriched in flagella and GSK3 associated with the axoneme in a phosphorylation-dependent manner. The level of active GSK3 correlated with flagellar length; early during flagellar regeneration, active GSK3 increased over basal levels. This increase in active GSK3 was rapidly lost within 30 min of regeneration as the level of active GSK3 decreased relative to the predeflagellation level. Taken together, these results suggest a possible role for GSK3 in regulating the assembly and length of flagella.  相似文献   

12.
Many studies have used velocity measurements, waveform analyses, and theoretical flagella models to investigate the establishment, maintenance, and function of flagella of the biflagellate green algae Chlamydomonas reinhardtii. We report the first direct measurement of Chlamydomonas flagellar swimming force. Using an optical trap ("optical tweezers") we detect a 75% decrease in swimming force between wild type (CC124) cells and mutants lacking outer flagellar dynein arms (oda1). This difference is consistent with previous estimates and validates the force measurement approach. To examine mechanisms underlying flagella organization and function, we deflagellated cells and examined force generation during flagellar regeneration. As expected, fully regenerated flagella are functionally equivalent to flagella of untreated wild type cells. However, analysis of swimming force vs. flagella length and the increase in force over regeneration time reveals intriguing patterns where increases in force do not always correspond with increases in length. These investigations of flagellar force, therefore, contribute to the understanding of Chlamydomonas motility, describe phenomena surrounding flagella regeneration, and demonstrate the advantages of the optical trapping technique in studies of cell motility.  相似文献   

13.
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.  相似文献   

14.
FLAGELLAR REGENERATION IN PROTOZOAN FLAGELLATES   总被引:44,自引:30,他引:14       下载免费PDF全文
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.  相似文献   

15.
The biflagellate somatic cells of Volvox carteri f. nagariensis lyengar exhibit an asymmetric pattern of flagellar development. Initiallt each somatic cell has two short (4 μm) flagella but after several hours one flagellum on each cell elongates unitl it reaches a length of 12 μm. Due to the regular arrangement of somatic cells in the Volvox spheroid it is apparent that the same flagellum on each somatic is the first to elongale. The asymmetric flagellar length is maintained for about 8 h after which the second flagellum on each somatic cell elongates. When the second flagellum attains the same length (12 μm) as the first flagellum, both flagella elongale at the same rate until reaching a final length of 22 μm. Experimental removal of somatic cell flagella results in their regeneration. Somatis cells regenerate both flagella simultaneously and full length flagella are produced in about 2 h. The intial rate of flagellar regeneration is about ten times faster than the intial rate of flagllar growth in development. Cycloheximide, an inhibitor of protein synthesis, has no effect on the initial rate of flagellar regeneration but the flagella produced in the presence of the drug are half the length of flagella produced in its absence. Somatic cells are able to regenerate flagella up to the time of α and β tubulin, the major structural proteins of the flagellar axoneme, and other cellular proteins.  相似文献   

16.
《The Journal of cell biology》1994,125(5):1119-1125
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.  相似文献   

17.
The effect of Ca(++) on the waveform of reactivated, isolated axonemes of chlamydomonas flagella was investigated. Flagella were detached and isolated by the dibucaine procedure and demembranated by treatment with the detergent Nonidet; the resulting axomenes lack the flagellar membrane and basal bodies. In Ca(++)-buffered reactivation solutions containing 10(-6) M or less free Ca(++), the axonemes beat with a highly asymmetrical, predominantly planar waveform that closely resembled that of in situ flagella of forward swimming cells. In solutions containing 10(-4) M Ca(++), the axonemes beat with a symmetrical waveform that was very similar to that of in situ flagella during backward swimming. In 10(-5) M Ca(++), the axonemes were predominantly quiescent, a state that appears to be closely associated with changes in axomenal waveform or direction of beat in many organisms. Experiments in which the concentrations of free Ca(++), not CaATP(--) complex were independently varied suggested that free Ca(++), not CaATP(--), was responsible for the observed changes. Analysis of the flagellar ATPases associated with the isolated axonemes and the nonidet- soluble membrane-matrix fraction obtained during preparation of the axonemes showed that the axonemes lacked the 3.0S Ca(++)-activated ATPase, almost all of which was recovered in the membrane-matrix fraction. These results indicate that free Ca(++) binds directly to an axonemal component to alter flagellar waveform, and that neither the 3.0S CaATPase nor the basal bodies are directly involved in this change.  相似文献   

18.
Chlamydomonas cells respond to certain environmental stimuli by shedding their flagella. Flagellar loss induces a rapid, transient increase in expression of a specific set of genes encoding flagellar proteins, and assembly of a new flagellar pair. While flagellar gene expression and initiation of flagellar outgrowth are normally tightly coupled to flagellar excision, our results demonstrate that these processes can be uncoupled by manipulating Ca2+ levels or calmodulin activity. In our experiments, wild-type cells were stimulated to excise their flagella using mechanical shearing, and at times after deflagellation, flagellar lengths were measured and flagellar mRNA abundance changes were determined by S1 nuclease protection analysis. When extracellular Ca2+ was lowered by addition of EGTA to cultures before excision, flagellar mRNA abundance changes and flagellar outgrowth were temporally uncoupled from flagellar excision. When extracellular Ca2+ was lowered immediately after excision or when calmodulin activity was inhibited with W-7, flagellar outgrowth was uncoupled from flagellar excision and flagellar mRNA abundance changes. Whenever events in the process of flagellar regeneration were temporally uncoupled, the magnitude of the flagellar mRNA abundance change was reduced. These results suggest that flagellar gene expression may be regulated by multiple signals generated from these events, and implicate Ca2+ as a factor in the mechanisms controlling flagellar regeneration.  相似文献   

19.
In order to elucidate mechanisms that control flagellar length of mature sperm, we studied in synchronous cell suspension cultures flagellar growth, tubulin pool, and tubulin synthesis in round spermatids of Xenopus laevis and the newt Cynops pyrrhogaster. The average final length of flagella in Xenopus round spermatids was 35 μm, almost the same length as that in mature sperm, whereas in the newt round spermatids, the length was 210 μm, almost half that of mature sperm. Kinetics of flagellar growth showed that the rate and period of flagellar growth in the newt spermatids were two to threefold those in Xenopus spermatids. The tubulin pool size in newt spermatids was estimated to be about 10-fold greater than that in Xenopus spermatids. But even if all of the pool was used for flagellar growth, it could support only about a seventh to a tenth of the flagellar length in mature sperm in either species. Thus, the possibility that the tubulin pool primarily determines flagellar length was excluded. Since the tubulin pool size did not change throughout the culture period, the possibility that the termination of flagellar growth is due to the exhaustion of the tubulin pool was also excluded. Tubulin synthesis declined over the culture period but continued in newt spermatids longer than in Xenopus spermatids. The period of flagellar elongation almost coincided with the period of tubulin synthesis. The amount of rRNA did not decrease, excluding the possibility that the decline of tubulin synthesis was due to cytoplasmic shedding which might result in the loss of ribosomes. Tubulin synthesis and the amount of rRNA in newt spermatids was more than threefold greater than that in Xenopus spermatids, which may explain the difference in growth rates of their flagella.  相似文献   

20.
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.  相似文献   

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