A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed.

In the present study, double immunofluorescence and immunoblot analysis have been used to show that centrosomes, isolated from Paracentrotus lividus sea urchin embryos at the first mitotic metaphase, contain the constitutive chaperone, heat-shock protein (HSP) 70. More specifically, we demonstrate that centrosomes contain only the HSP70-d isoform, which is one of the four isoforms identified in P. lividus. We also provide evidence that p34(cell division control kinase-2) and t complex polypeptide-1 (TCP-1) alpha, a subunit of the TCP-1 complex, are localized on the centrosomes. Furthermore, inhibition of TCP-1 in vivo, via microinjecting an anti-(TCP-1 alpha) antibody into P. lividus eggs before fertilization, either impaired mitosis or induced severe malformations in more than 50% of embryos. In addition, we have isolated the whole mitotic apparatus and shown that HSP70 localizes on the fibres of spindles and asters, and binds them in an ATP-dependent manner. These observations suggest that HSP70 has a chaperone role in assisting the TCP-1 complex in tubulin folding, when localized on centrosomes, and during the assembling and disassembling of the mitotic apparatus, when localized on the fibres of spindles and asters.

Constitutive hsp70 is essential to mitosis during early cleavage of Paracentrotus lividus embryos: the blockage of constitutive hsp70 impairs mitosis.

Localization of constitutive hsp70 in eggs and early embryos of sea urchin Paracentrotus lividus is shown by means of in situ immunostaining. An accumulation of this protein is shown in the mitotic structures (asters, spindles and centrosomes). Microinjection of anti-hsp70 antibodies into eggs causes impairment of formation of mitotic structures and of cell division. This impairment goes from a complete mitotic block, to irregular mitotic apparatus formation with irregular cleavage, depending upon the antibody concentration. The localization of hsp70 after antibody microinjection is also described. Blockage of mitotic apparatus formation by nocodazole also blocks the concentration of hsp70 molecules observed in nontreated eggs. That the constitutive hsp70 plays a role in sea urchin mitosis is indicated.

Deciliation: A stressful event for Paracentrotus lividus embryos.

In this report, by using mono- and two-dimensional electrophoretic analysis, we demonstrate that deciliation on sea urchin embryos induces a stress response. Deciliation indeed causes not only the activation of ciliary subroutine, but also a transient decrease of bulk protein synthesis. This decrease is in agreement with our previous results on heat shock response in sea urchin, although deciliation does not induce the expression of the same main hsp set. We were able to characterize one main deciliation-stress protein of 40 kDa whose expression is transiently induced by deciliation and whose localisation is likely to be nuclear.

Effect of the IMPase inhibitor L690,330 on sea urchin development.

A variety of concentrations of the IMPase inhibitor L690,330 were added to sea urchin embryos. Immediate arrest of development was obtained for concentrations from 7.5 mm on. Concentrations lower than 3.5 mm permitted gastrulation but inhibited skeletogenesis and disturbed elongation along the animal-vegetal axis. The latter results are similar to those obtained by counteracting lithium effect with myoinositol, which are suggested to be due to partial relief of IMPase inhibition.

Apoptosis in sea urchin embryos.

It is demonstrated by DNA electrophoresis analysis, morphological observations and TdT in situ reaction, that Paracentrotus embryos if treated with TPA plus heat undergo an apoptotic reaction. Indication is also obtained that non treated embryos undergo spontaneous apoptosis at the early pluteus stage, especially in the districts of arms and intestine. The possible meaning of this latter observation is discussed.

Sea urchin HSF activity in vitro and in transgenic embryos.

Evidence is provided for the presence at the physiological temperature of 20 degrees C of a heat shock transcriptor factor, HSF, in the nuclei of P.lividus embryos. This HSF is able to specifically bind in vitro the heat shock element, HSE, of the promoter of the hsp70 gene i.v., as suggested by DNA-protein binding reactions and DNAse I protection assays. Upon heat-shock, at the temperature of 31 degrees C, its ability to bind the HSE units becomes much higher. The HSF activated by heat-shock drives in vivo the transcription of the beta-galactosidase reporter gene in transgenic sea urchin gastrulae. An ATF-like transcription factor, widely described in other organisms but not at all in sea urchins, is also present in the nuclear extracts and is able to bind the consensus individuated in the hsp70 i.v. gene promoter.

Sea urchin mitochondrial matrix contains a 56-kDa chaperonine-like protein.

Paracentrotus lividus mitochondrial matrix contains a constitutive hsp of 56-KDa which cross reacts with a serum anti-hsp-60 chaperonine from yeast mitochondria. The localization of hsps preexisting or newly synthesized in different subcellular fractions of gastrula embryos is also analyzed by two-dimensional electrophoresis.

Identification and characterization of a constitutive HSP75 in sea urchin embryos.

An antiserum against a hsp of the 70-kDa family was prepared, by means of a fusion protein, which was able to detect a constitutive 75-kDa hsc in the sea urchin P. lividus. This hsc was present both during oogenesis and at all developmental stages. A two-dimensional electrophoresis has revealed four isolectric forms of this 75-kDa hsc. The amino acid sequence of the fragment used to prepare the anti-hsp70 antibodies revealed a 43% identity with the corresponding part of sea urchin sperm receptor, and in mature eggs a brighter immunofluorescence was seen all around the cell cortex where the receptor for sea urchin sperm is localized. In oocytes the hsp75 was localized in the cytoplasms but not in the nuclei. In the embryos a higher hsp75 concentration was found in the portion facing the lumen of the cells which invaginate at gastrulation.

Effect of retinoic acid and valproate on sea urchin development.

The effect of valproate and retinoic acid on Paracentrotus lividus development and fertilization is reported. Retinoic acid at concentrations between 5 microM and 7 microM causes severe disturbance of development and at 10 microM complete embryo degeneration if added in the period from fertilization on. Concentrations up to 7 microM are uneffective if retinoic acid addition is started 16 h after fertilization. Valproate at concentrations between 1 mM and 5 mM causes severe disturbances of development if added from 5 min after fertilization on. Concentrations of 20 mM are lethal to all embryos. If the drug is added 16 h after fertilization no effect is observed up to the concentration of 10 mM. The effect of valproate is specific, because its isomere 2-en at 10 mM has very little effect on development. Both drugs affect fertilization only at higher concentrations: more than 1 microM for retinoic acid and more than 10 mM for valproate.

Activation by heat shock of hsp70 gene transcription in sea urchin embryos.

We had previously shown that P. lividus embryos subjected to heat shock are unable to synthesize proteins of the hsp70 family at a detectable level before the hatching blastula stage. We show here that this is not due to the inability to synthesize the hsp70 mRNAs, which are detectable following heat shock also in early stages, although in much lower amounts per embryo than in later stages. These mRNAs are also translated, as judged by the facts that they are present in the polysomal pellet, and that they are translatable in a cell free system. As to the question of the amount of hsp70 RNAs per nucleus, we conclude that this is also higher in later than in earlier stages. The presence of hsp70 mRNAs is already detectable after heating at 4 centigrades above 20 and their amount increases with the increase of temperature in the range between 24 degrees C and 28 degrees C.

Effect of doxorubicin and phenytoin on sea urchin development.

The effect of doxorubicin and phenytoin on Paracentrotus lividus development and fertilization is reported. Doxorubicin at concentrations between 1.4 and 1.8 microM causes severe disturbances of development and at 2.0 microM, complete embryonic degeneration, if added in the period from fertilization on. Concentrations up to 20 microM are ineffective on development if the treatment is started at 12 h after fertilization. Phenytoin at concentrations between 4.0 and 7.0 microM causes severe disturbances of development, if added from 5 min after fertilization on. Concentrations of 10 microM are lethal to all embryos. Again, if the drug is added at 12 h after fertilization, no effect is observed up to the concentration of 20 microM. Both drugs are ineffective on the fertilization process up to the concentration of 20 microM for doxorubicin and of 200 microM for phenytoin.

Achievement of thermotolerance through hsps phosphorylation in sea urchin embryos.

TPA treatment of sea urchin embryos is able to induce thermotolerance. Evidence is provided that TPA treatment induces phosphorylation of a constitutive stress protein of 38 KDa.

Two-dimensional electrophoretic analysis of stress proteins in Paracentrotus lividus.

The synthesis of stress proteins in Paracentrotus lividus embryos has been analysed by two-dimensional electrophoresis following either heat shock or ZnSO4 administration. As already shown for the 70-KDa hsp (heat shock proteins) a non responsive period, that is before hatching, exists followed by a responsive period in which up to 16 hsps are synthesized after heat shock, 7 of which are also synthesized after ZnSO4 treatment. Some of them pre-existed the stress, but their synthesis increases following the stress. Only few of them accumulate in amounts detectable by silver staining. The synthesis of all these proteins depends upon RNA synthesis.

Stress proteins by zinc ions in sea urchin embryos.

In Paracentrotus lividus embryos, treatment with zinc ions induces the synthesis of the two major stress proteins with the same molecular weight as those induced by heat shock. The developmental stages responsive to zinc ion treatment are the same as those responsive to heat shock. However, zinc treatment induces a longer lasting synthesis of the stress proteins, and, unlike heat shock, does not induce thermotolerance and does not inhibit synthesis of the bulk proteins.

Reconstitution of membranes and embryonic development in dissociated blastula cells of the sea urchin by reinsertion of aggregation-promoting membrane proteins extracted with butanol.

Blastula embryos of the sea urchin Paracentrotus lividus, when dissociated into single cells by exposure to Ca2+- and Mg2+-free sea water, reassociate spontaneously to form aggregates capable of development to the final larval form (pluteus). This aggregation is prevented by Fab fragments obtained by immunization with purified membranes from blastula embryos. The inhibition was reversed by soluble proteins extracted with butanol from purified membranes or from intact cells. These extracts also strongly stimulated the rate of reaggregation of dissociated cells in the absence of Fab fragments. Exposure of dissociated cells to 2.5% (vol/vol) butanol removed completely the protein(s) responsible for reaggregation of the cells without impairing their viability. Reaggregation and embryonic development were completely restored to the extracted cells by readdition of the proteins extracted from either membranes or cells. Extracted cells from Paracentrotus could be reconstituted with proteins from Arbacia.