Gérard Coffe

Cold-treated centrosome: isolation of centrosomes from mitotic sea urchin eggs, production of an anticentrosomal antibody, and novel ultrastructural imaging.

A novel isolation of centrosomes is described and it was used to both generate a centrosome-specific monoclonal antibody and to image with high-resolution low-voltage scanning electron microscopy the surface details of the isolated centrosome. At first mitotic prometaphase, sea urchin zygotes are chilled on ice overnight. While most of the microtubules disassemble, the mitotic centrosomes collapse into aggregated masses. These centrosomes have been isolated, and used to generate a monoclonal antibody, designated 4D2, which is reactive with interphase and mitotic centrosomes. 4D2 staining of centrosomes is similar, but not identical, to that of other centrosomal antibodies like Ah6 and 5051. Centrosomal material is detected as a compact sphere after cold treatment; upon recovery the sphere expands and undergoes the shape changes previously described [Mazia et al., 1987: J. Cell Biol. 105:206a] to eventually reorganize a normal mitotic apparatus.

Purification, in vitro reassembly, and preliminary sequence analysis of epiplasmins, the major constituent of the membrane skeleton of Paramecium.

The epiplasmic layer, a continuous rigid granulo-fibrillar sheet directly subtending the surface membranes of Paramecium, is one of the outermost of the various cytoskeletal networks that compose it cortex. We have previously shown that the epiplasm consists of a set of 30 to 50 protein bands on SDS-PAGE in the range 50 to 33 kDa, the epiplasmins. We report a purification procedure for the set of epiplasmic proteins, a description of their physicochemical and reassembly properties, and a preliminary characterization of their sequence. The conditions for solubilization of the epiplasm and for in vitro reassembly of its purified constituents ar described. Reassembly of the entire set of proteins and of some (but not all) subsets are shown to yield filamentous aggregates. Microsequences of two purified bands of epiplasmins reveal a striking amino acid sequence consisting of heptad repeats of only three main amino acids, P, V, and Q. These repeats were confirmed by DNA sequencing of polymerase chain reaction products. The motif is QPVQ-h, in which h is a hydrophobic residue. This may constitute the core of the epiplasmin sequence and, in view of the tendency of such a sequence to form a coiled-coil, may account for the remarkable self-aggregation properties of epiplasmins.

Association between microtubules and Golgi vesicles isolated from rat parotid glands

We report an isolation procedure of trans-Golgi vesicles (GVs) from rat parotid glands. Various organelle markers were used, particularly galactosyl transferase as a trans-Golgi marker, to test the purity of the GV fraction. A quantitative in vitro binding assay between microtubules and GVs is described. The vesicles were incubated with taxol-induced microtubules, layered between 50% and 43% sucrose cushions and subjected to centrifugation. Unlike free microtubules which were sedimented, the GV-bound microtubules co-migrated upward with GVs. Quantification of these bound microtubules was carried out by densitometric scanning of Coomassie blue-stained gels. The association between microtubules and GVs followed a saturation curve, with a plateau value of 20 micrograms of microtubule protein bound to 500 micrograms of GV fraction. The half-saturation of the GV sites was obtained with a microtubule concentration of 20 micrograms/ml. Electron microscopy of negatively stained re-floated material showed numerous microtubule-vesicle complexes. Coating of microtubules with an excess of brain microtubule-associated proteins (MAPs) abolished binding. In the absence of exogenous microtubules, we showed that the GV fraction was already interacting with a class of endogenous rat parotid microtubules. This class of colcemid and cold-stable microtubules represents 10-20% of the total tubulin content of the parotid cell.

Distribution of a centrosomal antigen during morphogenesis in the ciliated protozoan Euplotes

Ciliates assemble basal bodies in great number at many stages of the life-cycle. In order to understand their assembly mechanisms, we screened a library of monoclonal antibodies directed against pericentriolar material. One of these antibodies, CTR210, was used previously to follow steps of this assembly process: in Paraurostyla, new basal bodies appear along a scaffold of linear structures recognized by this antibody. The very unusual behavior of this antigen deserved confirmation in other species. In the present study, we show by immunofluorescence that, in another phylogenetically very distant species, Euplotes, basal bodies are assembled in the same pathway during division. In addition, this antibody recognizes a filamentous ring located at the division furrow and linking many basal body assemblages. By cell fractionation and cytoskeletal extraction, we obtained fractions enriched in basal bodies and associated material. Such fractions still display a high complexity in protein composition. These fractions were used to characterize the main target of the antibody as a doublet of 45 kDa. These results confirm previous results in terms of functionality of the protein recognized by the antibody, but raise new questions in terms of the assignment of the recognized protein to the HSP70 family as hypothesized previously.

Tubulin dynamics during the cytoplasmic cohesiveness cycle in artificially activated sea urchin eggs

Sedimentation studies and [3H]colchicine-binding assays have demonstrated a relationship between the cytoplasmic cohesiveness cycles and the changes in tubulin organization in Paracentrotus lividus eggs activated by 2.5 mM procaine. The same amount of tubulin (20-25% of the total egg tubulin) is involved in these cyclic process and appears to undergo polymerization and depolymerization cycles. Electron microscopy studies reveal that the microtubules formed during these cytoplasmic cohesiveness cycles are under a particulate form which is sedimentable at low speed. Activation experiments carried out in the presence of cytochalasin B (CB) show that the increase in the cytoplasmic cohesiveness is highly reduced while tubulin polymerization and depolymerization cycles and pronuclear centration are not affected. Although tubulin or actin polymerization can be independently triggered in procaine-activated eggs, the increase in cytoplasmic cohesiveness requires the polymerization of both proteins. However, the cytoplasmic cohesiveness cycles appear to be regulated by tubulin polymerization and depolymerization cycles.

A network of 2–4 nm filaments found in sea urchin smooth muscle: Protein constituents and in situ localization☆

In this report the coisolation of two proteins from sea urchin smooth muscle of apparent molecular weights (Mr) 54 and 56 kD respectively, as determined on SDS-PAGE, is described. Like the intermediate filament proteins, these two proteins are insoluble in high ionic strength buffer solution. On two-dimensional gel electrophoresis and by immunological methods it is shown that these proteins are not related (by these criteria) to rat smooth muscle desmin (54 kD) or vimentin (56 kD). Furthermore, in conditions where both desmin and vimentin assemble in vitro into 10 nm filaments, the sea urchin smooth muscle proteins do not assemble into filaments. Ultrastructural studies on the sea urchin smooth muscle cell show that the thin and thick filaments organization resembles that described in the vertebrate smooth muscle. However, instead of 10 nm filaments, a network of filaments, 2-4 nm in diameter, is revealed, upon removal of the thin and thick filaments by 0.6 M KCl treatment. By indirect immunofluorescence microscopy, and in particular by immunocytochemical electron microscopy studies on the sea urchin smooth muscle cell, it is shown that the antibodies raised against both 54 and 56 kD proteins appear to specifically label these 2-4 nm filaments. These findings indicate that both the 54 and 56 kD proteins might be constituents of this category of filaments. The possible significance of this new cytoskeletal element, that we have named echinonematin filaments, is discussed.

Dual effect of procaine in sea urchin eggs: Inducer and inhibitor of microtubule assembly☆

An increase in the amount of cytoplasmic filamentous structures (cytoplasmic matrix and aster) which were recovered after hexylene glycol/Triton X-100 treatment of sea urchin eggs (Paracentrotus lividus) activated by 0.2-2.5 mM procaine was observed. At higher activator concentrations, an opposite effect was observed and formation of these cytoplasmic structures was inhibited in the presence of 10 mM procaine. This inhibitory effect was reversed by diluting the drug in the incubation medium. DNase I inhibition assays on egg homogenates which were performed at different time points of the activation process, show that the same amount of actin was induced to polymerize in eggs activated either by 2.5 or 10 mM procaine. However, colchicine-binding assays on the 100 000 g particulate fractions of these homogenates show that in eggs activated by 10 mM procaine, in contrast to those activated by 2.5 mM, tubulin polymerization was inhibited and microtubules were disassembled. These results show that the dual effect of procaine in the organization of the egg cytoskeleton appears to be related to its effect on the state of tubulin.

Actin purification from a gel of rat brain extracts

Actin, 99% pure, has been recovered from rat brain with a high yield (greater than 15 mg/100 g brain). We have shown that: 1. a low ionic strength extract from rat brain tissue is capable of giving rise to a gel; 2. actin is the main gel component and its proportion is one order of magnitude higher than in the original extract; 3. actin can be isolated from this extract by a three-step procedure involving gelation, dissociation of the gel in 0.6 M KCl, followed by one or two depolymerization-polymerization cycles.

Chemical reactivity of the tyrosyl residues in yeast hexokinase: Properties of the nitroenzyme

Abstract Of the 15 tyrosyl residues per subunit of yeast hexokinase A (ATP : d -hexose 6-phosphotransferase, EC 2.7.1.1) two are more accessible to chemical modification with cyanuryl fluoride at pH 9.7 (0°C), or with tetranitromethane at pH 8.0 (0° or 22°C) while only one is modified with N -acetylimidazol at pH 7.5 (22°C). In presence of 0.2 M glucose, only one of the two accessible tyrosyl residues is modified by nitration with tetranitromethane at pH 8.0 (22°C). The nitration of this tyrosyl residue induces dissociation of the protein to its monomeric forms and results in a loss of 90% of enzyme activity. The partially active nitroenzyme has the same pH activity profile as the native enzyme. Furthermore, K m values for glucose and MgATP are not significatively affected. Moreover, neither the negative cooperativity with respect to MgATP nor the citrate activation effect is affected by the modification of the tyrosyl residue and dissociation of the protein. However the burst-type slow transient in the reaction progress curve is abolished by the nitration process. These results show that negative cooperativity and the citrate activation effect are not as strictly dependent on the conformational state of the protein as the burst-type slow transient. Furthermore, the large decrease in enzyme activity due to the nitration of the tyrosyl group is related simultaneously to the dissociation of the protein and probably also to the perturbation of the conformation of the enzyme active center region.