Localization of cytoskeletal proteins in Pneumocystis carinii by immuno-electron microscopy

Pneumocystis carinii causes serious pulmonary infection in immunosuppressed patients. This study was undertaken to observe the cytoskeletal proteins of P. carinii by immuno-electron microscopy. P. carinii infection was experimentally induced by immunosuppression of Sprague-Dawley rats for seven weeks, and their lungs were used for the observations of this study. The gold particles localized actin, tropomyosin, and tubulin. The actin was irregularly scattered in the cytoplasm of the trophic forms but was much more concentrated in the inner space of the cell wall of the cystic forms called the inner electron-lucent layer. No significant amount of tropomyosin was observed in either trophic forms or cystic forms. The tubulin was distributed along the peripheral cytoplasm and filopodia of both the trophic and cystic forms rather than in the inner side of the cytoplasm. Particularly, in the cystic forms, the amount of tubulin was increased and located mainly in the inner electron-lucent layer of the cell wall where the actin was concentrated as well. The results of this study showed that the cell wall of P. carinii cystic forms is a structure whose inner side is rich in actin and tubulin. The location of the actin and tubulin in P. carinii suggests that the main role of these proteins is an involvement in the protection of cystic forms from the outside environment by maintaining rigidity of the cystic forms.

Spectrophotometric titration of phenoxyl groups of sheep brain tubulin.

Spectrophotometric titration of phenoxyl group of sheep brain tubulin carried out in 6M guanidine hydrochloride indicated 36 tyrosine residues per dimer of tubulin. A plot of log alpha/(1-alpha) versus pH showed that 26 residues titrated with apparent pK of 10.4 and 10 residues titrated with apparent pK of 11.0. The high pK value of tyrosine could be attributed to the possibility that the molecules containing tyrosine residues were not completely utilised as these residues could have been partially shielded from the solvent. Alternatively, they could have proximal negative charges.

Acetylated alpha-tubulin in Trypanosoma cruzi: immunocytochemical localization

We have used monoclonal antibodies specific for acetylated and non-acetylated alpha-tubulin to localize microtubules containing acetylated alpha-tubulin in all developmental forms of the life cycle of Trypanosoma cruzi. This was demonstrated using immunofluorescence and by transmission electron microscopy of thin sections, negative stained cells, and replicas of whole Triton X-100 extracted cells immunolabeled with antibody-gold complex. The antibody specific for acetylated alpha-tubulin (6-11B-1) binds to the flagellar, as well as to the sub-pellicular microtubules. The extent of labeling of the sub-pellicular microtubules with the monoclonal antibody recognized alpha-acetylated tubulin was smaller than that observed with the antibody which recognizes all tubulin isoforms. In relation to the developmental forms, the extent of labeling of the microtubules with antibody 6-11B-1 was larger in epimastigote and trypomastigote than in amastigote forms. Incubation of the parasites for 1 h at 0 degrees C or in the presence of either colchicine or vinblastine did not interfere with the sub-pellicular microtubules. These observations, in agreement with those reported for Trypanosoma brucei brucei (Schneider et al., 1987; Schulze et al., 1987; Sasse per cent Gull, 1988) indicate that the sub-pellicular microtubules of trypanosomatids represent stable microtubules containing acetylated alpha-tubulin (or the alpha 3-tubulin isotype)

An ELISA method for quantitation of tubulin using poly-1-lysine coated microtiter plates.

A new, sensitive and convenient ELISA method has been developed for quantitation of tubulin using poly-1-lysine (PLL) coated multiwell microtiter plates. Binding of tubulin to untreated plastic surface of microtiter plates was extremely poor. Coating of wells with PLL enhanced the binding and facilitated quantitation by ELISA. Binding of tubulin was followed by stepwise additions of rabbit anti-tubulin IgG, HRP-conjugated goat anti-rabbit IgG and colour reagent. The method has been successfully applied to quantitate the tubulin content of extracts from rat brain and liver as evident from the excellent correlation of the results with those obtained from 3H-colchicine binding assay. The detection limit is as low as 5 ng, which is relatively better than that of the previous RIA methods. The ELISA method does not involve the use of any radioactive compound and all reagents required for this assay are commercially available.