Tubulin synthesis in sea urchin embryos II. Ciliary a tubulin derives from the unfertilized egg

Bibring, Thomas; Baxandall, Jane

doi:10.1016/s0012-1606(81)80014-0

Cited by 28 publications

(9 citation statements)

References 30 publications

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“…The finding that ATPase activity regenerates faster than AK activity supports the idea that embryos contain pools of dynein that can be utilized for ciliary regeneration [Bibring and Baxandall, 1981;Foltz and Asai, 1990;Stephens, 1977Stephens, , 1989Stephens, , 1995Stephens, , 1997. A large pool of tubulin [Bibring and Baxandall, 1981] and dynein [Foltz and Asai, 1990] are present in embryos that can be utilized for regeneration of cilia.…”

Section: Regeneration Of Atpase and Sp-ak Activitysupporting

confidence: 56%

Adenylate kinase in sea urchin embryonic cilia

Kinukawa

Vacquier

2007

Cell Motil. Cytoskeleton

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Sea urchin embryos swim by ciliary movement. Hypertonic shock causes deciliation and loss of motility. Within 2-4 h, cilia regenerate and the embryos swim again. Regeneration of cilia occurs multiple times. The adenylate kinase (AK) activity of isolated cilia was studied. A 130-kDa Sp-AK isozyme, present in sperm flagella, is also present in embryonic cilia. AK activity is responsible for approximately 93% of nonmitochondrial ATP regeneration from ADP in embryonic cilia. This is unlike sea urchin sperm flagella, where approximately 31% of the nonmitochondrial ATP regeneration is from the 130-kDa Sp-AK isozyme and approximately 69% from the flagellar creatine kinase (Sp-CK). Embryos were deciliated 1-3 times and after a 2-h period of regeneration the major ciliary axonemal proteins such as the tubulins appeared constant in amount. However, a moderate decrease in ATPase activity, and a large decrease of total AK activity, were measured. The decrease in AK activity paralleled the decrease in embryo swimming velocity. Embryos were deciliated once and cilia regeneration followed for 4 h. ATPase activity recovered to control levels by 3 h, but AK activity and swimming velocity remained lower than in controls. Detergent solubility data and kinetic experiments indicate that, in addition to the 130-kDa Sp-AK, there is at least one additional AK isozyme in embryonic cilia. Analysis of the S. purpuratus genome indicates five AK isozymes in addition to the 130-kDa Sp-AK isozyme. Decreased swimming velocity of embryos with regenerated cilia suggests that regenerated cilia are not as functionally perfect as naturally grown cilia.

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Section: Regeneration Of Atpase and Sp-ak Activitysupporting

confidence: 56%

Adenylate kinase in sea urchin embryonic cilia

Kinukawa

Vacquier

2007

Cell Motil. Cytoskeleton

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“…Most of the proteins sedimented near the top of the gradient, apparently after being dissociated from larger structures by the high salt concentration. The peak at 22 S (fractions 18-26) contained several intense high molecular weight protein bands and a set of polypeptides in the range of [24][25][26][27][28][29][30][31][32][33][34][35] (the lanes containing the latter proteins are labeled "P"). The data suggest that this set ofproteins sedimented as a unit (at -19 S) separate from both the high molecular size -WI W,l proteins, which seem to sediment more rapidly (at 22 S), and a doublet of 21-and 24-kDa proteins that sediments more slowly.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, the results indicate that the prosome must be synthesized during oogenesis and stored in the mature egg. Such storage of stable maternal components is well known and is the case for ribosomes (21), some chromatin proteins (24), and embryonic cilia tubulin (25).…”

Section: Discussionmentioning

confidence: 99%

Sea urchin prosome: characterization and changes during development.

Akhayat¹,

De²,

Infante³

1987

Proc. Natl. Acad. Sci. U.S.A.

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A cytoplasmic particle displaying properties in common with a structure present in duck erythroblasts, termed the prosome, has been isolated from eggs and embryos of two species of sea urchin. This particle was partially purified by sedimentation in sucrose gradients containing 0.5 M KCI, and some of its physical properties and its behavior during early development were determined. The prosome sediments between 16 and 19 S and has a buoyant density of 1.30 g/cm3 in Cs2SO4 gradients. Biochemically, the particle is characterized as 20-25 polypeptides of molecular size 24-35 kDa with about 10 small RNAs. A monoclonal antibody directed against the 27-kDa protein of duck erythroblast prosome recognizes a 27-kDa protein of the sea urchin prosome. We have used this protein, as representative of the prosome, to immunologically determine the level and the subcellular localization of the particle during development. Immunoblotting and cellular fractionation studies show that the 27-kDa prosome polypeptide is present almost entirely in the postribosomal supernatant of unfertilized egg lysates. After fertilization and during early development, the total amount of 27-kDa protein per embryo remains constant, but the amount in the postribosomal supernatant decreases; there is a concomitant increase in the level of the 27-kDa protein in a rapidly sedimenting, particulate fraction containing nuclei. Immunofluorescence studies further show that the 27-kDa protein is located mainly in the cytoplasm of eggs and two-cell embryos. The subcellular location of the prosome, therefore, appears to change during development. In vivo labeling experiments have failed to detect the synthesis of either the prosome proteins or RNAs in eggs and embryos up to 48 hr of development, suggesting that this cytoplasmic particle is not synthesized de novo in early embryogenesis and thus is metabolically stable. The prosome is thus a normal cellular constituent of the sea urchin and is most likely synthesized during oogenesis and stored in the unfertilized egg.

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“…This pool provides most of the tubulins for the first mitotic spindle (13) and the cilia of the blastula (14). Despite this excess, tubulin synthesis increases after fertilization, roughly in parallel with overall protein synthesis (6,15).…”

Section: -9)mentioning

confidence: 99%

Multiple polymorphic alpha- and beta-tubulin mRNAs are present in sea urchin eggs.

Alexandraki¹,

Ruderman²

1985

Proc. Natl. Acad. Sci. U.S.A.

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Multiple a-and -tubulin RNAs were found in the mature unfertilized eggs of the sea urchin Lytechinus pictus. The a-tubulin RNAs were polymorphic in number, size, and relative amounts in the eggs of different females. Five to seven different size classes [1.75-4.2 kilobases (kb)] were detected on RNA gel blots. All egg preparations contained variable amounts of 1.8-and 2.25-kb (-tubulin RNAs, and a few of them contained an additional 2.9-kb (-tubulin RNA. The total amount of a-tubulin RNA did not always parallel that of 3-tubulin RNA. A portion of all of the various a-and (-tubulin RNAs were polyadenylylated. RNase H digestions ruled out the possibility that some of these RNAs represented a single transcript bearing different lengths of 3' poly(A). One class of a-tubulin RNAs (2.4-2.65 kb) was reduced to 2 kb by RNase H, suggesting the presence of internal oligo(A) regions. All of the egg (3-tubulin RNAs sedimented as free ribonucleoprotein particles. Only a small portion of the 1.75-to 3.6-kb atubulin RNAs, but most of the 4.2-kb a-tubulin RNA, were found on polysomes before fertilization. In the 30-min embryo, small amounts of each of the various a-and j-tubulin RNAs were recruited onto polysomes. Thus, each of the multiple polymorphic a-and ,B-tubulin RNAs in the egg represent translationally competent mRNA. rhe sea urchin egg contains large stockpiles of structural proteins and enzymes that help sustain the rapid cleavage divisions of the early embryo (1-6). Little is known about the kinetics of synthesis of these proteins during oogenesis. In the first readily available stage, the mature unfertilized egg, the overall rate of protein synthesis is low (reviewed in refs. 7-9).a-and ,3-tubulins synthesized during oogenesis (10) make up 1%-5% of the egg protein (11,12). This pool provides most of the tubulins for the first mitotic spindle (13) MATERIALS AND METHODS Preparation of Egg RNA. Lytechinus pictus (Pacific Biomarine, Venice, CA) eggs collected and treated as described (19) were lysed in 0.35 M NaCl/1 mM EDTA/10 mM Tris'HCl, pH 8/2% NaDodSO4/7 M urea. Total nucleic acids were purified by phenol/chloroform/isoamyl alcohol (25:24:1) extractions. Isolation of testis RNA, and fractionation of RNA on oligo(dT)-cellulose into poly(A)+ and poly(A)-RNAs have been described (19). Treatment of RNA with RNase H. RNA samples were treated with RNase H as described by the supplier (Bethesda Research Laboratories).Polysome Gradients. Eggs and embryos were homogenized in 0.3 M glycine/250 mM KCl/3 mM magnesium chloride/40 mM Hepes, pH 7.3, and supernatants were centrifuged at 12,000 x g on 11 ml of 15%-40% (wt/vol) sucrose gradients (made in 0.5 M KCl/6 mM magnesium chloride/i mM EDTA/10 mM Hepes, pH 7.4) for 95 min at 40,000 rpm in a SW41 rotor (28,29). In some experiments, the RNA was released from polysomes prior to centrifugation by treatment with 2 mM puromycin dihydrochloride or 30 mM EDTA (30, 31).RNA Gel Blot Analysis. RNA gel blotting and hybridization were carried out as described (29 (20). The non-cod...

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Tubulin synthesis in sea urchin embryos II. Ciliary a tubulin derives from the unfertilized egg

Cited by 28 publications

References 30 publications

Adenylate kinase in sea urchin embryonic cilia

Adenylate kinase in sea urchin embryonic cilia

Sea urchin prosome: characterization and changes during development.

Multiple polymorphic alpha- and beta-tubulin mRNAs are present in sea urchin eggs.

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