Appearance of the flagellate phenotype in populations of Naegleria amebae

Fulton, Chandler; Dingle, Allan D.

doi:10.1016/0012-1606(67)90012-7

Cited by 90 publications

(78 citation statements)

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“…Cell suspensions, 25 ml at approximately 107 cells per ml, were incubated at 25.0°C in 250-ml Erlenmeyer flasks in a reciprocating water bath at 100 strokes per min (23). The appearance of flagellates was monitored by fixing cells in Lugol iodine and counting flagellates with a phase-contrast microscope (13). The completeness and timing of all differentiations were followed by allowing one flask of cells to form swimming flagellates.…”

Section: Methodsmentioning

confidence: 99%

“…In contrast to C. reinhardtii, N. gruberi amebae have no morphological trace of basal bodies (14), flagellar axonemes (9), or other parts of the flagellar apparatus (24,25,36), yet they can differentiate into swimming flagellates in 2 h (10,11,13). Under carefully * Corresponding author.…”

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confidence: 99%

“…controlled laboratory conditions, the differentiation is synchronous and complete with more than 95% of the cells forming flagella (13).…”

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Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amebae into flagellates.

Lee

Walsh

1988

Mol. Cell. Biol.

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The nuclear run-on technique was used to measure the rate of transcription of flagellar genes during the differentiation of Naegleria gruberi amebae into flagellates. Synthesis of mRNAs for the axonemal proteins aand l-tubulin and flagellar calmodulin, as wel as a coordinately regulated poly(A)+ RNA that codes for an unidentified protein, showed transient increases averaging 22-fold. The rate of synthesis of two poly(A)+ RNAs common to amebae and flagellates was low until the transcription of the flagellar genes began to decline, at which time synthesis of the RNAs found in amebae increased 3-to 10-fold. The observed changes in the rate of transcription can account quantitatively for the 20-fold increase in flagellar mRNA concentration during the differentiation. The data for the flageilar calmodulin gene demonstrate transcriptional regulation for a nontubulin axonemal protein. The data also demonstrate at least two programs of transcriptional regulation during the differentiation and raise the intriguing possibility that some significant fraction of the nearly 200 different proteins of the flagellar axoneme is transcriptionally regulated during the 1 h it takes N. grubeni amebae to form visible flagella.Axonemes of eucaryotic flagella are composed of nearly 200 different proteins (28). Thus, neglecting proteins associated with basal bodies and the various rootlet structures found in most cells, the formation of cilia or flagella requires the synthesis and assembly of a minimum of 200 gene products. This complexity is particularly notable because some cells can assemble flagella quite rapidly.The best-studied example of flagellum formation is the biflagellated green alga Chlamydomonas reinhardtii. Most studies of flagellum formation in C. reinhardtii have taken advantage of the fact that the cells are readily deflagellated to examine the regeneration of flagella (e.g., see references 27, 32, 33, and 39). Regeneration involves both the assembly of preexisting flagellar proteins, which can produce about 50% of the original flagellar length, and the synthesis of new protein (33). Transient increases in a number of mRNAs coding for flagellar proteins have been documented during C. reinhardi flagellar regeneration (34), but changes in only two of these RNAs have been studied at the transcriptional level. Changes in the level of mRNAs for the a and ,B subunits of tubulin were found to be due, in part, to changes in the rate of their synthesis and, in part, to changes in tubulin mRNA stability (2,19). No data are available to suggest how proteins that are exclusively flagellar are regulated (C. reinhardtii contains only one form of ,-tubulin which presumably must be used in cytoplasmic and mitotic microtubules as well as flagella) (40). It is also of interest to ask whether the regulatory mechanisms governing regeneration of flagella in a normally flagellated cell would apply to a cell forming flagella de novo.The amebo-flagellate Naegleria gruberi provides an opportunity to examine this question. In contrast to C. rei...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amebae into flagellates.

Lee

Walsh

1988

Mol. Cell. Biol.

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“…Dilute detergent (0 .005% Nonidet P-40) was present during fixation to allow more efficient penetration of antibodies . Incubation of amoebae fixed immediately upon initiation of differentiation with antibodies to the rootlet protein, followed by fluorescein-conju- 12 Densitometer tracing of purified rootlet protein separated electrophoretically by the Weber-Osborn 5% SDS polyacrylamide phosphate gel system . The gel was stained with Coomassie Brilliant Blue and scanned at 560 nm with a Gilford microdensitometer (Gifford Instrument Laboratories Inc ., Oberlin, Ohio) .…”

Section: Rootlet Localization In Vivomentioning

confidence: 99%

Isolation, ultrastructure, and protein composition of the flagellar rootlet of Naegleria gruberi.

Larson¹,

Dingle²

1981

The Journal of Cell Biology

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Attached to the basal bodies of Naegleria gruberi flagellates is a striated rootlet or rhizoplast . The rootlet-basal body complex has been isolated by Triton X-100 lysis of deflagellated cells and differential centrifugation through a 25% glycerol medium . Rootlets isolated from mature flagellates are -13 p,m long but vary from 8 to 15 ftm in length : they taper at both ends from a maximum width of -0 .25 p,m in the vicinity of the basal bodies . They are highly stable during isolation but can be solubilized by urea, high salt, low pH, or detergent (Sarkosyl) . Partial dissociation of rootlets with 1 M urea reveals that they are composed of filaments, -5 nm diameter, associated in linear fashion to yield the characteristic 21-nm cross-banded appearance . Differential solubilization of rootlets and their associated contaminants allowed identification of a major rootlet protein, comprising at least 50% of any purified rootlet preparation, with an apparent subunit molecular weight of 170,000. The localization of rootlets in situ by indirect immunofluorescence using a specific antibody directed against the purified rootlet protein demonstrated unequivocally that this 170,000-dalton protein is an organelle component.The flagella of eukaryotic cells are generally found as part of a flagellar apparatus-an interconnected assemblage of basal body, flagellum, and a striated rootlet fiber or rhizoplast . Likewise, cilia, basal bodies, and one or more rootlets often combine to constitute what could be called a "ciliary apparatus" in ciliated cells . Although flagellated and ciliated cells lacking rootlets are known, and even common (e .g., sperm cells, certain mammalian ciliated epithelia), some form of rootlet structure is attached to the basal bodies of most cilia and flagella (7,8,30) . Despite intense current interest in the structure, function, and morphogenesis of flagella and their basal bodies, virtually no attention has been paid to the other major component of the flagellar or ciliary apparatus, the rootlet. References to flagellar rootlets have most often been restricted to ultrastructural descriptions and to speculation about possible roles in motility (5,9,15,16,19,26,28,34,38) .Electron micrographs of rootlets generally reveal bundles of 4-to 5-nm-diameter filaments aggregated in a paracrystalline array to produce fibrous, cross-striated organelles reminiscent of collagen fibrils (9, 26, 28, 37) . The cross-striations form one major repeating unit that may be further divided into -13 intraperiod sub-bands (28, 38) . Reported widths for this major repeat unit range from 12 to 78 nm (34,35) . Though most authors describe a constant banding pattern, at least three reports have commented upon the variations in rootlet period-424 icity (15,32,34) and have inferred a possible physiological role for the altered states . The idea that the rootlet is a contractile element of the flagellar apparatus which may play an active role in modifying the flagellar beat is supported by the following : the recent demonst...

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“…However, considering the position of hydra on the evolutionary scale, the analogy to protozoa which are often directly affected by environmental stimuli to undergo differentiation (e.g. Fulton and Dingle, 1967;Fulton, 1972) may be equally appropriate. In this case, the influence would be directly on the stem cell.…”

Section: (B) Hormonal Influence On Sex Determinationmentioning

confidence: 99%