Using a monoclonal antibody generated against striated muscle troponin T, we previously noted the presence of crossreactive components in smooth muscle and non-muscle cells. Since the presence of troponin T in tissues other than striated muscle is controversial, we sought to establish the nature of the crossreaction and to determine the extent of its occurrence. For this study, indirect immunofluorescence microscopy and immunoblot analyses were performed. Crossreactive material was found in diverse cells from the animal, plant and fungal kingdoms. On the basis of morphological distributions, both microtubule-associated and non-microtubule-associated components were revealed. Microtubule-associated components of animal cell lines included a 35×103Mr protein, similar in electrophoretic mobility to skeletal troponin T (37×103Mr). Reactive components of comparable mobility were observed in immunoblots of brain and cerebellar homogenates. Filamentous staining was observed in a variety of mammalian cells in culture and in cells of vertebrate tissues. Chick cerebellar tissue showed reactions in the neurites of the molecular layer and granule cell bodies. In the plant kingdom, examination of the onion root-tip cells indicated an association of crossreactive components with interphase cortical microtubules, preprophase bands, the mitotic spindle and phragmoplast microtubules. In the fungal kingdom, both interphase and mitotic spindle microtubules in a cellular slime mould were reactive. Nonmicrotubule-associated components were observed in the centrosphere regions of mitotic seaurchin eggs, in mitotic and interphase plasmodia of Physarum polycephalum, and in trichocysts and basal bodies of Paramecium tetraurelia. In all systems examined, the troponin T crossreactive components were located in regions or on structures of possible Ca2+ or calmodulin activity, suggesting a possible functional similarity to troponin T.

The calcium control of striated muscle contraction is regulated through troponintropomyosin interactions. Troponin is a complex of three subunits designated Tn-I, Tn-Cand Tn-T (Greaser & Gergely, 1971). Tn-C is the calcium binding component that interacts with Tn-I to relieve the inhibition of the latter on the ATPase activity of actomyosin. However, it is only in the presence of all three troponin subunits that the system becomes sensitive to calcium ions. In this respect Tn-T, which has a high affinity for the calcium-insensitive tropomyosin as well as for the Ca2+-binding Tn-C, represents a critical regulatory component of the troponin complex (Ohtsuki, 1980).

Many attempts have been made to compare the regulation of cytoplasmic actin-myosin interaction with that of the highly differentiated striated muscle counterpart. However, in the non-muscle systems, the presence of a true muscletype troponin complex is still controversial and even the function of cytoplasmic tropomyosin is not well understood. Fine et al. (1973) have shown that nonmuscle tropomyosin isolated from brain tissue has a peptide map similar to muscle tropomyosin and binds to actin. It is also able to substitute for muscle tropomyosin in a vertebrate skeletal muscle regulatory system. In the absence of a muscle-type troponin complex in non-muscle cells, the functional capacity of tropomyosin in the Ca2+ regulatory process is apparently not used. Instead, a structural role has been suggested in view of its preferential association with stress fibres (Lazarides, 1976).

Previously, we reported the production of a monoclonal antibody generated against striated muscle troponin T that crossreacted with troponin T in all muscle types (Lim et al. 1984) and that crossreacted with components in smooth muscle and non-muscle cells (Lim et al. 1985). In this study, we report the immunochemical crossreaction of this anti-troponin T with components in animal, plant and fungal kingdoms. Its widespread occurrence in cells of diverse phylogenetic origin implies the conservation of a biologically important determinant. On the basis of morphological distribution as determined by indirect immunofluorescence microscopy, two groups of anti-troponin T crossreactive components were observed : those associated with microtubules and those not associated with microtubules. The localization of the crossreactive components in regions or structures of possible Ca2+ or calmodulin activity suggests that an epitope shared with troponin T may be of general regulatory significance in non-muscle cells.

Monoclonal antibodies

The production and initial characterization of the mouse monoclonal antibody (IgM) directed against rabbit skeletal troponin T have been described elsewhere (Lim et al. 1984, 1985). The rat monoclonal antibody (IgG) against tubulin was a gift from Dr J. Kilmartin (Kilmartin et al. 1982).

Indirect immunofluorescence microscopy

Cell lines

Mouse 3T3 fibroblasts, 356 human foreskin fibroblasts, rat kangaroo kidney (PtKj) cells, neuroblastoma (N2A) cells and Chinese hamster ovary (CHO) cells were grown on glass coverslips in Ham’s F-10 medium supplemented with 10% foetal bovine serum (FBS). HeLa cells were grown in F-10 supplemented with 10% horse serum. Cells plated on coverslips were rinsed twice in phosphate-buffered saline (PBS), pH7·4, and then fixed in 1% glutaraldehyde, 0·2% Nonidet P-40 (NP-40), 10 mM-EGTA in PBS, pH 7·4, for 20 min at room temperature. After three washes in PBS, the cells were treated with lmgml-1 sodium borohydride for 1 h. After three washes in PBS, the cells were incubated for l h with the appropriate primary antibody (45% (NH4)2SO4-cut hybridoma supernatant, 2·3 mg ml-1) at 37°C. This was followed by three washes in PBS to free the cells of excess primary antibody. After incubation with the secondary antibody (FITC-conjugated anti-mouse IgM) for 0·5 h at 37°C, the coverslips were rinsed and then mounted in a medium containing lmgml-1 p-phenylenediamine as an anti-bleaching agent (Sammak & Borisy, unpublished data).

For double-label indirect immunofluorescence microscopy incubation of each primary antibody was followed by its corresponding secondary antibody. For example, anti-troponin T was followed by affinity-purified goat anti-mouse IgM (μ-chain-specific, FITC-conjugated; Cappel Labs), after which the second primary antibody, anti-tubulin, was followed by goat anti-rat IgG (gamma-chainspecific, RITC-conjugated; Cappel Labs). Cells were examined with a Zeiss Universal epifluorescence microscope equipped with narrow band fluorescein and rhodamine excitation filters.

In control experiments, primary antibody from hybridomas was substituted by PBS or cell supernatants from hybridoma secreting antibodies to unrelated antigens (gift from Dr Patricia Witt, Molecular Biology Laboratory, University of Wisconsin). In double-label indirect immunofluorescence control experiments, the goat anti-mouse IgM (μ-chain-specific) FITC was reacted with the rat IgG anti-tubulin (YL 1/2). No reaction was observed.

Paraffin sections of nervous tissue

The cerebellum was isolated from a newborn chick, fixed in 95% ethanol/5% ethanol/5% acetic acid and prepared for paraffin embedment and indirect immunofluorescence microscopy according to the procedure of Saint-Marie (1962). Sections of about 4pm were cut and stained.

Plant cell preparation

Seeds of onion (Allium) were germinated in the dark at room temperature on moist filter paper. When the roots were about 1 cm long, the apical 1-4 mm was cut and fixed for 1 h in freshly prepared 3·7% formaldehyde in PBS, pH7·4, containing 50mM-EGTA. After treatment with 1 % cellulysin (Sigma) in 0·4M-mannitol and 5mM-EGTA (Wick et al. 1981), the root tips were again rinsed in buffer and squashed between coverslips to release individual cells.

Dictyostelium discoideum

An axenic strain of the cellular slime mould (A×3) was a gift from the laboratory of Dr Randall Dimond (Department of Bacteriology, University of Wisconsin). After the density of the cells was determined under the phase-contrast microscope, the cells were allowed to settle onto polylysine-coated coverslips for about 30 min, then fixed with 3·7% formaldehyde in PBS, pH 7·4. After permeabilization with both methanol and acetone, the cells were processed for immunofluorescence microscopy as described by White et al. (1983).

Physarum polycephalum

Samples of P. polycephalum were provided by Eileen Paul from the laboratory of Dr William Dove (McArdle Laboratory for Cancer Research, University of Wisconsin). Smears of macroplasmodia from the slime mould were prepared for indirect immunofluorescence microscopy according to Havercroft & Gull (1983).

Sea-urchin

(Strongylocentrotus purpuratus) eggs. Sea-urchin egg samples were a gift from Dr Ryoko Kuriyama (Department of Anatomy, University of Minnesota). Fertilized eggs were allowed to develop through first mitosis. After checking the density of the eggs under the phasecontrast microscope, they were allowed to settle onto polylysine-coated coverslips and then fixed in 2% glutaraldehyde in 0’4M-sodium acetate buffer, pH 6·0, with 1% Triton X-100 and 50 mM-EGTA (Asnes & Schroeder, 1979). The coverslips were then routinely processed for indirect immunofluorescence microscopy.

Paramecium tetraurelia

Samples of Paramecia were provided by Andrew Levin from the laboratory of Dr David Nelson (Department of Biochemistry, University of Wisconsin). The cells were deciliated in 100 mM-MnCL and fixed with 3·7 % paraformaldehyde in 60 mM-PIPES, 25 mM-HEPES, 10mM-EGTA and 2mM-MgC12 (PHEM), pH 6·9. After they were allowed to settle onto polylysine-coated coverslips, the cells wére processed as usual for indirect immunofluorescence microscopy.

SDS–polyacrylamide gel electrophoresis and immunoblotting

Preparation of gel samples: HeLa cell homogenate was prepared by addition of hot sample buffer (Laemmli, 1970) to the culture dish. HeLa cytoskeletons were prepared by stabilizing cells with 10μgml-1 taxol for 15min, followed by treatment (90s) with lysis buffer containing 10mM-PIPES, pH 6’9, 0’2% NP-40, 5μgml-1 taxol and 1 mM-phenylmethylsulphonyl fluoride (PMSF). After solubilization in hot sample buffer all samples were further lysed by three 30-s pulses from a Heat Systems Sonifier (Branson Ultrasonics, Plainview, NY), setting 3. Samples were then applied to polyacrylamide gels (Laemmli, 1970). Preparations of myofibrils (Wang, 1982) and chick cerebellum were homogenized at 0°C in 0·1 M-Tris-HC1 buffer, pH7-3, containing l-OmM-PMSF. Samples were then resuspended in hot sample buffer and boiled for 15 min.

After electrophoretic separation, the polypeptides were transferred electrophoretically onto nitrocellulose paper (Towbin et al. 1979). The blots were stained for protein in 0·1% Amido Black in 45% methanol and 10% acetic acid, while duplicate blots were treated with a blocking solution (20mM-Tris, 0·9% NaCl, 10% horse serum) and then reacted with a 1:1000 dilution of anti-troponin T ascitic fluid (5·6 mg ml-1 IgM). To visualize specific antigen-antibody reactions, the enzyme immunoassay kit and protocol from BIO-RAD laboratories was used (BIORAD Immuno-blot AGR-HRP Assay Kit). Controls for the immunoblot experiments included substitution of the blocking buffer for primary antibody and non-immune mouse IgM.

Widespread occurrence of an anti-troponin T microtubule-associated protein

Mammalian cells

Several mammalian cell lines were examined, including 3T3 mouse fibroblasts, 356 human foreskin fibroblasts, PtK1 cells, CHO cells and HeLa cells. Both interphase and mitotic cells reacted with the antibody as revealed by indirect immunofluorescence microscopy in all cell lines. In interphase cells (Fig. 1) brightly stained punctate networks of cytoplasmic filaments were observed. These filaments converged on a perinuclear focus. The anti-troponin T (Anti-Tn-T) also identified crossreactive components on midbodies and mitotic spindles of dividing cells (Fig. 2). These morphological patterns suggested microtubule distribution. To verify this, double-label indirect immunofluorescence microscopy was performed, which permitted the simultaneous visualization of two antigens in the same cell. The filamentous pattern recognized by the anti-troponin T was spatially coincident with that of microtubules, as indicated by anti-tubulin. Higher-magnification micrographs (Figs 3, 4) also show very clearly the punctate nature of the anti-troponin T staining pattern at this dilution (Fig. 3) as compared to the linear pattern of the anti-tubulin staining (Fig. 4). The fact that the anti-troponin T decorated only microtubules and no other cytoskeletal elements (e.g. stress fibres, intermediate filaments) was also apparent.

Fig. 1.

Interphase fibroblasts (356 human foreskin fibroblast) reacted with the anti-TnT reveal staining of a cytoplasmic filamentous array with a perinuclear focus. Bar, 10 μm.

Fig. 1.

Interphase fibroblasts (356 human foreskin fibroblast) reacted with the anti-TnT reveal staining of a cytoplasmic filamentous array with a perinuclear focus. Bar, 10 μm.

Fig. 2.

Mitotic fibroblasts (356 human foreskin fibroblast) reacted with anti-Tn-T indicate reaction with the mitotic apparatus. Bar, 10 μM.

Fig. 2.

Mitotic fibroblasts (356 human foreskin fibroblast) reacted with anti-Tn-T indicate reaction with the mitotic apparatus. Bar, 10 μM.

Fig. 3.

Double-label indirect immunofluorescence (356 human foreskin fibroblast) with anti-Tn-T followed by FITC-conjugated anti-mouse IgM and anti-tubulin followed by RITC-conjugated anti-rat IgG. The punctate filamentous array described by the anti-Tn-T is spatially coincident with the microtubule pattern indicated by the anti-tubulin. Bar, 8μm.

Fig. 3.

Double-label indirect immunofluorescence (356 human foreskin fibroblast) with anti-Tn-T followed by FITC-conjugated anti-mouse IgM and anti-tubulin followed by RITC-conjugated anti-rat IgG. The punctate filamentous array described by the anti-Tn-T is spatially coincident with the microtubule pattern indicated by the anti-tubulin. Bar, 8μm.

Fig. 4.

Double-label indirect immunofluorescence (356 human foreskin fibroblast) with anti-Tn-T followed by FITC-conjugated anti-mouse IgM and anti-tubulin followed by RITC-conjugated anti-rat IgG. The punctate filamentous array described by the anti-Tn-T is spatially coincident with the microtubule pattern indicated by the anti-tubulin. Bar, 8μm.

Fig. 4.

Double-label indirect immunofluorescence (356 human foreskin fibroblast) with anti-Tn-T followed by FITC-conjugated anti-mouse IgM and anti-tubulin followed by RITC-conjugated anti-rat IgG. The punctate filamentous array described by the anti-Tn-T is spatially coincident with the microtubule pattern indicated by the anti-tubulin. Bar, 8μm.

To obtain information concerning the molecular weight of the anti-troponin T crossreactive species, immunoblots containing samples of HeLa whole cell homogenate (Fig. 5, lane 3) and HeLa cytoskeletons (Fig. 5, lane 4) were treated with anti-troponin T. (Compare with the reactive troponin T band in the myofibril preparation, lane 2.) A strong reaction with a 35 × 103Mr band was detected in both HeLa cell samples. The retention of the reactive 35×103Mr band in the cell cytoskeleton fraction (Fig. 5, lane 4) confirmed our indirect immunofluorescence observations that the anti-troponin T crossreactive species was associated with a component of the cytoskeleton, most probably microtubules. A faint reaction was sometimes observed at the 55 × 103Mr band of both HeLa samples. Immunoblots of two-dimensional gels (data not shown) indicated this to be due to a crossreaction with beta-tubulin (see Discussion). However, no reaction was observed with the 55×103.Wr tubulin band from the brain microtubule protein preparation in lane 5, even though strong reactions were apparent with some high molecular weight polypeptides (see Discussion).

Fig. 5.

Double-label indirect immunofluorescence (356 human foreskin fibroblast) with anti-Tn-T followed by FITC-conjugated anti-mouse IgM and anti-tubulin followed by RITC-conjugated anti-rat IgG. The punctate filamentous array described by the anti-Tn-T is spatially coincident with the microtubule pattern indicated by the anti-tubulin. Bar, 8μm.

Fig. 5.

Double-label indirect immunofluorescence (356 human foreskin fibroblast) with anti-Tn-T followed by FITC-conjugated anti-mouse IgM and anti-tubulin followed by RITC-conjugated anti-rat IgG. The punctate filamentous array described by the anti-Tn-T is spatially coincident with the microtubule pattern indicated by the anti-tubulin. Bar, 8μm.

Nerve cells and tissues

Paraffin sections through the thoracic regions of 5-day chick embryos revealed crossreaction with a major non-muscle tissue mass. In addition to the reaction with the somitic regions (skeletal muscle precursor) and forming myocardium, cellular components of the neural tube as well as fibre tracts were intensely stained (Figs 6, 7). Since the resolution of tissue sections (particularly 4-μm paraffin sections) was inadequate to determine whether the crossreactive component observed in nerve cells was microtubule-associated, we decided to examine nerve cells in culture. Double immunofluorescence of neuroblastoma (N2A) revealed a co-localization of the anti-troponin T with microtubules (Figs 8, 9). Immunofluorescence of primary cultures of dissociated parasympathetic ganglion cells from rat (gift from Dr Philippa Claude, Primate Center, University of Wisconsin) also revealed filamentous staining in the neurites (Figs 10, 11). Intense staining was also observed in the cell bodies. However, as these regions are quite thick, it was not possible to observe any detail in the staining pattern.

Fig. 6.

Indirect immunofluorescence using anti-Tn-T on paraffin sections through thoracic region of 5-day chick embryo (transverse sections). Fig. 6 shows that in addition to reaction with the somitic regions (s), intense staining is seen at the neural tube (n) as well as the forming spinal nerve (sn). Higher magnification (Fig. 7) shows reaction with fibre tract at floor of neural tube. Fig. 6, bar, 100μm; Fig. 7, bar, 2μm.

Fig. 6.

Indirect immunofluorescence using anti-Tn-T on paraffin sections through thoracic region of 5-day chick embryo (transverse sections). Fig. 6 shows that in addition to reaction with the somitic regions (s), intense staining is seen at the neural tube (n) as well as the forming spinal nerve (sn). Higher magnification (Fig. 7) shows reaction with fibre tract at floor of neural tube. Fig. 6, bar, 100μm; Fig. 7, bar, 2μm.

Fig. 7.

Indirect immunofluorescence using anti-Tn-T on paraffin sections through thoracic region of 5-day chick embryo (transverse sections). Fig. 6 shows that in addition to reaction with the somitic regions (s), intense staining is seen at the neural tube (n) as well as the forming spinal nerve (sn). Higher magnification (Fig. 7) shows reaction with fibre tract at floor of neural tube. Fig. 6, bar, 100μm; Fig. 7, bar, 2μm.

Fig. 7.

Indirect immunofluorescence using anti-Tn-T on paraffin sections through thoracic region of 5-day chick embryo (transverse sections). Fig. 6 shows that in addition to reaction with the somitic regions (s), intense staining is seen at the neural tube (n) as well as the forming spinal nerve (sn). Higher magnification (Fig. 7) shows reaction with fibre tract at floor of neural tube. Fig. 6, bar, 100μm; Fig. 7, bar, 2μm.

Fig. 8.

Double-label immunofluorescence of neuroblastoma (N2A) cells using anti-Tn-T visualized in the fluorescein channel (Fig. 8) and anti-tubulin visualized in the rhodamine channel (Fig. 9). Again, co-localization of the anti-Tn-T crossreactive species with microtubules is apparent. Bar, 10μm.

Fig. 8.

Double-label immunofluorescence of neuroblastoma (N2A) cells using anti-Tn-T visualized in the fluorescein channel (Fig. 8) and anti-tubulin visualized in the rhodamine channel (Fig. 9). Again, co-localization of the anti-Tn-T crossreactive species with microtubules is apparent. Bar, 10μm.

Fig. 9.

Double-label immunofluorescence of neuroblastoma (N2A) cells using anti-Tn-T visualized in the fluorescein channel (Fig. 8) and anti-tubulin visualized in the rhodamine channel (Fig. 9). Again, co-localization of the anti-Tn-T crossreactive species with microtubules is apparent. Bar, 10μm.

Fig. 9.

Double-label immunofluorescence of neuroblastoma (N2A) cells using anti-Tn-T visualized in the fluorescein channel (Fig. 8) and anti-tubulin visualized in the rhodamine channel (Fig. 9). Again, co-localization of the anti-Tn-T crossreactive species with microtubules is apparent. Bar, 10μm.

Fig. 10.

Indirect immunofluorescence on primary cultures of dissociated parasympathetic ganglion cells from rat. anti-Tn-T staining reveals intense reaction with cell bodies (c) as well as filamentous arrays in neurite. Bar, 8 μm.

Fig. 10.

Indirect immunofluorescence on primary cultures of dissociated parasympathetic ganglion cells from rat. anti-Tn-T staining reveals intense reaction with cell bodies (c) as well as filamentous arrays in neurite. Bar, 8 μm.

Fig. 11.

Indirect immunofluorescence on primary cultures of dissociated parasympathetic ganglion cells from rat. anti-Tn-T staining reveals intense reaction with cell bodies (c) as well as filamentous arrays in neurite. Bar, 8 μm.

Fig. 11.

Indirect immunofluorescence on primary cultures of dissociated parasympathetic ganglion cells from rat. anti-Tn-T staining reveals intense reaction with cell bodies (c) as well as filamentous arrays in neurite. Bar, 8 μm.

Paraffin sections of newly hatched chick brain and cerebellum also showed an abundance of the crossreactive component. In the cerebellum (Figs 1217) the pattern was most striking. The anti-troponin T crossreacted with specific regions, including the granular layer (Figs 12, 16) and the neurites of the molecular layer (Figs 12, 13). The dendrites of the Purkinje cells were prominently reactive (Figs 14, 15). No staining was observed in the internal white matter (Fig. 17).

Fig. 12.

Indirect immunofluorescent micrographs of paraffin-embedded sections of newly hatched chick cerebellum. Anti-Tn-T is seen in both the molecular layer (m) and granular layer (g). In the molecular layer, staining of neurites as well as some cell bodies is apparent (Fig. 13). Purkinje cells (P) and their dendrites are visibly reactive (Figs 14, 15). Granule cells stain intensely (Fig. 16), while no reaction is seen in the internal white matter except for that with some granule cells (Fig. 17). Fig. 12, bar, 45 μm; Fig. 13, bar, 22 um; Figs 14–17, bar, 19μm.

Fig. 12.

Indirect immunofluorescent micrographs of paraffin-embedded sections of newly hatched chick cerebellum. Anti-Tn-T is seen in both the molecular layer (m) and granular layer (g). In the molecular layer, staining of neurites as well as some cell bodies is apparent (Fig. 13). Purkinje cells (P) and their dendrites are visibly reactive (Figs 14, 15). Granule cells stain intensely (Fig. 16), while no reaction is seen in the internal white matter except for that with some granule cells (Fig. 17). Fig. 12, bar, 45 μm; Fig. 13, bar, 22 um; Figs 14–17, bar, 19μm.

Fig. 13.

Indirect immunofluorescent micrographs of paraffin-embedded sections of newly hatched chick cerebellum. Anti-Tn-T is seen in both the molecular layer (m) and granular layer (g). In the molecular layer, staining of neurites as well as some cell bodies is apparent (Fig. 13). Purkinje cells (P) and their dendrites are visibly reactive (Figs 14, 15). Granule cells stain intensely (Fig. 16), while no reaction is seen in the internal white matter except for that with some granule cells (Fig. 17). Fig. 12, bar, 45 μm; Fig. 13, bar, 22 um; Figs 14–17, bar, 19μm.

Fig. 13.

Indirect immunofluorescent micrographs of paraffin-embedded sections of newly hatched chick cerebellum. Anti-Tn-T is seen in both the molecular layer (m) and granular layer (g). In the molecular layer, staining of neurites as well as some cell bodies is apparent (Fig. 13). Purkinje cells (P) and their dendrites are visibly reactive (Figs 14, 15). Granule cells stain intensely (Fig. 16), while no reaction is seen in the internal white matter except for that with some granule cells (Fig. 17). Fig. 12, bar, 45 μm; Fig. 13, bar, 22 um; Figs 14–17, bar, 19μm.

Fig. 14.

Indirect immunofluorescent micrographs of paraffin-embedded sections of newly hatched chick cerebellum. Anti-Tn-T is seen in both the molecular layer (m) and granular layer (g). In the molecular layer, staining of neurites as well as some cell bodies is apparent (Fig. 13). Purkinje cells (P) and their dendrites are visibly reactive (Figs 14, 15). Granule cells stain intensely (Fig. 16), while no reaction is seen in the internal white matter except for that with some granule cells (Fig. 17). Fig. 12, bar, 45 μm; Fig. 13, bar, 22 um; Figs 14–17, bar, 19μm.

Fig. 14.

Indirect immunofluorescent micrographs of paraffin-embedded sections of newly hatched chick cerebellum. Anti-Tn-T is seen in both the molecular layer (m) and granular layer (g). In the molecular layer, staining of neurites as well as some cell bodies is apparent (Fig. 13). Purkinje cells (P) and their dendrites are visibly reactive (Figs 14, 15). Granule cells stain intensely (Fig. 16), while no reaction is seen in the internal white matter except for that with some granule cells (Fig. 17). Fig. 12, bar, 45 μm; Fig. 13, bar, 22 um; Figs 14–17, bar, 19μm.

Fig. 15.

Indirect immunofluorescent micrographs of paraffin-embedded sections of newly hatched chick cerebellum. Anti-Tn-T is seen in both the molecular layer (m) and granular layer (g). In the molecular layer, staining of neurites as well as some cell bodies is apparent (Fig. 13). Purkinje cells (P) and their dendrites are visibly reactive (Figs 14, 15). Granule cells stain intensely (Fig. 16), while no reaction is seen in the internal white matter except for that with some granule cells (Fig. 17). Fig. 12, bar, 45 μm; Fig. 13, bar, 22 um; Figs 14–17, bar, 19μm.

Fig. 15.

Indirect immunofluorescent micrographs of paraffin-embedded sections of newly hatched chick cerebellum. Anti-Tn-T is seen in both the molecular layer (m) and granular layer (g). In the molecular layer, staining of neurites as well as some cell bodies is apparent (Fig. 13). Purkinje cells (P) and their dendrites are visibly reactive (Figs 14, 15). Granule cells stain intensely (Fig. 16), while no reaction is seen in the internal white matter except for that with some granule cells (Fig. 17). Fig. 12, bar, 45 μm; Fig. 13, bar, 22 um; Figs 14–17, bar, 19μm.

Fig. 16.

Indirect immunofluorescent micrographs of paraffin-embedded sections of newly hatched chick cerebellum. Anti-Tn-T is seen in both the molecular layer (m) and granular layer (g). In the molecular layer, staining of neurites as well as some cell bodies is apparent (Fig. 13). Purkinje cells (P) and their dendrites are visibly reactive (Figs 14, 15). Granule cells stain intensely (Fig. 16), while no reaction is seen in the internal white matter except for that with some granule cells (Fig. 17). Fig. 12, bar, 45 μm; Fig. 13, bar, 22 um; Figs 14–17, bar, 19μm.

Fig. 16.

Indirect immunofluorescent micrographs of paraffin-embedded sections of newly hatched chick cerebellum. Anti-Tn-T is seen in both the molecular layer (m) and granular layer (g). In the molecular layer, staining of neurites as well as some cell bodies is apparent (Fig. 13). Purkinje cells (P) and their dendrites are visibly reactive (Figs 14, 15). Granule cells stain intensely (Fig. 16), while no reaction is seen in the internal white matter except for that with some granule cells (Fig. 17). Fig. 12, bar, 45 μm; Fig. 13, bar, 22 um; Figs 14–17, bar, 19μm.

Fig. 17.

Indirect immunofluorescent micrographs of paraffin-embedded sections of newly hatched chick cerebellum. Anti-Tn-T is seen in both the molecular layer (m) and granular layer (g). In the molecular layer, staining of neurites as well as some cell bodies is apparent (Fig. 13). Purkinje cells (P) and their dendrites are visibly reactive (Figs 14, 15). Granule cells stain intensely (Fig. 16), while no reaction is seen in the internal white matter except for that with some granule cells (Fig. 17). Fig. 12, bar, 45 μm; Fig. 13, bar, 22 um; Figs 14–17, bar, 19μm.

Fig. 17.

Indirect immunofluorescent micrographs of paraffin-embedded sections of newly hatched chick cerebellum. Anti-Tn-T is seen in both the molecular layer (m) and granular layer (g). In the molecular layer, staining of neurites as well as some cell bodies is apparent (Fig. 13). Purkinje cells (P) and their dendrites are visibly reactive (Figs 14, 15). Granule cells stain intensely (Fig. 16), while no reaction is seen in the internal white matter except for that with some granule cells (Fig. 17). Fig. 12, bar, 45 μm; Fig. 13, bar, 22 um; Figs 14–17, bar, 19μm.

Immunoblots of brain microtubule protein (Fig. 5) indicated a crossreaction with a high molecular weight species identified as MAP IA (unpublished data). While certain features of the anti-troponin T crossreaction were similar to that reported by Bloom et al. (1984) for MAP IA (i.e. the reaction of the Purkinje cells, Figs 14, 15) the reaction in the granule cell layer and the lack of reaction in the internal white matter differed from the reported MAP IA distribution. This suggested the possibility that the pattern seen in the cerebellum was due to more than one molecule. This was borne out by our results in immunoblots of brain and cerebellum. As in brain, two reactive bands were observed in the cerebellar homogenate (Fig. 18): bands corresponding to MAP I and a lower molecular weight band at about 35(×103)Mr. This lower molecular weight band was not present in our immunoblots of brain MTP preparations, indicating that it was ‘cycled off’ during the procedure, unlike the other microtubule-associated proteins (MAP 1, MAP 2 and tau proteins), which remain associated.

Fig. 18.

immunoblot of chick cerebellar homogenate reacted with the anti-Tn-T. A. Amido Black-stained nitrocellulose to visualize transferred proteins; B, transferred proteins reacted with anti-Tn-T; lanes 1, myofibril preparation; lanes 2, chick cerebellar homogenate. Note crossreaction with a protein band with mobility close to that of Tn-T (compare lanes 1, 2, in B). High molecular weight components, most probably MAP 1 (see Fig. 9) are also reactive; 10% acrylamide gels.

Fig. 18.

immunoblot of chick cerebellar homogenate reacted with the anti-Tn-T. A. Amido Black-stained nitrocellulose to visualize transferred proteins; B, transferred proteins reacted with anti-Tn-T; lanes 1, myofibril preparation; lanes 2, chick cerebellar homogenate. Note crossreaction with a protein band with mobility close to that of Tn-T (compare lanes 1, 2, in B). High molecular weight components, most probably MAP 1 (see Fig. 9) are also reactive; 10% acrylamide gels.

Plant cells

Examination of meristematic cells of onion (Allium) root revealed a pattern of anti-troponin T crossreaction very similar to that described by others for microtubule distribution. In interphase cells (Fig. 19), cortical arrays of well-aligned microtubules were observed while the nuclear region was non-reactive. In cells that were preparing to divide, reaction with the pre-prophase band was seen (Fig. 20). This band of microtubules, which spans the circumference of the cell cortex, predicts the site and plane of subsequent cytokinesis (Wick et al. 1981). In mitotic cells (Fig. 21) the anti-troponin T reacted with the spindle fibres as well as the polar cap regions onto which the microtubules converge. At late anaphase anti-troponin T staining of the phragmoplast region was evident. The co-localization of the antitroponin T crossreactive components with microtubules of the onio.i cells was further confirmed by double-label indirect immunofluorescence (data not shown).

Fig. 19.

Indirect immunofluorescence of meristematic onion (Allium) root-tip cells.

The various known categories of microtubular arrays are all reactive with the anti-Tn-T ; the cortical arrays of relatively well aligned microtubules in interphase cells (Fig. 19), the pre-prophase band, which predicts the site and plane of future cytokinesis (Fig. 20), the mitotic spindle (Fig. 21) and the phragmoplast (p) (Fig. 22). Bar, 8μm.

Fig. 19.

Indirect immunofluorescence of meristematic onion (Allium) root-tip cells.

The various known categories of microtubular arrays are all reactive with the anti-Tn-T ; the cortical arrays of relatively well aligned microtubules in interphase cells (Fig. 19), the pre-prophase band, which predicts the site and plane of future cytokinesis (Fig. 20), the mitotic spindle (Fig. 21) and the phragmoplast (p) (Fig. 22). Bar, 8μm.

Fig. 20.

Indirect immunofluorescence of meristematic onion (Allium) root-tip cells.

The various known categories of microtubular arrays are all reactive with the anti-Tn-T ; the cortical arrays of relatively well aligned microtubules in interphase cells (Fig. 19), the pre-prophase band, which predicts the site and plane of future cytokinesis (Fig. 20), the mitotic spindle (Fig. 21) and the phragmoplast (p) (Fig. 22). Bar, 8μm.

Fig. 20.

Indirect immunofluorescence of meristematic onion (Allium) root-tip cells.

The various known categories of microtubular arrays are all reactive with the anti-Tn-T ; the cortical arrays of relatively well aligned microtubules in interphase cells (Fig. 19), the pre-prophase band, which predicts the site and plane of future cytokinesis (Fig. 20), the mitotic spindle (Fig. 21) and the phragmoplast (p) (Fig. 22). Bar, 8μm.

Fig. 21.

Indirect immunofluorescence of meristematic onion (Allium) root-tip cells.

The various known categories of microtubular arrays are all reactive with the anti-Tn-T ; the cortical arrays of relatively well aligned microtubules in interphase cells (Fig. 19), the pre-prophase band, which predicts the site and plane of future cytokinesis (Fig. 20), the mitotic spindle (Fig. 21) and the phragmoplast (p) (Fig. 22). Bar, 8μm.

Fig. 21.

Indirect immunofluorescence of meristematic onion (Allium) root-tip cells.

The various known categories of microtubular arrays are all reactive with the anti-Tn-T ; the cortical arrays of relatively well aligned microtubules in interphase cells (Fig. 19), the pre-prophase band, which predicts the site and plane of future cytokinesis (Fig. 20), the mitotic spindle (Fig. 21) and the phragmoplast (p) (Fig. 22). Bar, 8μm.

Fig. 22.

Indirect immunofluorescence of meristematic onion (Allium) root-tip cells.

The various known categories of microtubular arrays are all reactive with the anti-Tn-T ; the cortical arrays of relatively well aligned microtubules in interphase cells (Fig. 19), the pre-prophase band, which predicts the site and plane of future cytokinesis (Fig. 20), the mitotic spindle (Fig. 21) and the phragmoplast (p) (Fig. 22). Bar, 8μm.

Fig. 22.

Indirect immunofluorescence of meristematic onion (Allium) root-tip cells.

The various known categories of microtubular arrays are all reactive with the anti-Tn-T ; the cortical arrays of relatively well aligned microtubules in interphase cells (Fig. 19), the pre-prophase band, which predicts the site and plane of future cytokinesis (Fig. 20), the mitotic spindle (Fig. 21) and the phragmoplast (p) (Fig. 22). Bar, 8μm.

Dictyostelium discoideum

The organization of the microtubules in the cellular slime mould has been previously visualized using antibodies against tubulin (Cappuccinelli et al. 1982). The anti-troponin T staining of an axenic strain (Ax3) revealed a pattern similar to that reported for anti-tubulin. The cells of this strain are often multinucleate. In interphase cells (Fig. 23) the antibody reacted with cytoplasmic microtubules that originated from distinct microtubule-organizing centres (nucleus-associated bodies). In mitotic cells (Figs 24, 25) the interphase network of microtubules was replaced by the intranuclear pole body. In early prophase the antibody reacted with the spindle pole bodies, which had duplicated and were now connected by short bundles of microtubules (Fig. 24). These increased in length as mitosis progressed (Fig. 25). At telophase cytoplasmic microtubules in the dividing daughter cells were again apparent (Fig. 26). In all instances the co-localization of the anti-troponin T staining with that of anti-tubulin was further confirmed by double-label indirect immunofluorescence in these cells (data not shown).

Fig. 23.

Indirect immunofluorescence micrographs of cellular slime mould D. discoideum reacted with anti-Tn-T. The cells of this axenic strain (Ax3) are often multinucleate. In interphase cells (Fig. 23) the antibody reacted with cytoplasmic microtubules, which originate from distinct organizing centres (nucleus associated bodies) to radiate in all directions; during mitosis (intranuclear), the spindle pole bodies connected by slim bundles of microtubules are reactive (Figs 24, 25). At late telophase, dividing daughter cells can be observed to regain the cytoplasmic microtubule arrays (Fig. 26). Fig. 23, bar, 5 urn; Figs 24–26, bar, 6μm.

Fig. 23.

Indirect immunofluorescence micrographs of cellular slime mould D. discoideum reacted with anti-Tn-T. The cells of this axenic strain (Ax3) are often multinucleate. In interphase cells (Fig. 23) the antibody reacted with cytoplasmic microtubules, which originate from distinct organizing centres (nucleus associated bodies) to radiate in all directions; during mitosis (intranuclear), the spindle pole bodies connected by slim bundles of microtubules are reactive (Figs 24, 25). At late telophase, dividing daughter cells can be observed to regain the cytoplasmic microtubule arrays (Fig. 26). Fig. 23, bar, 5 urn; Figs 24–26, bar, 6μm.

Fig. 24.

Indirect immunofluorescence micrographs of cellular slime mould D. discoideum reacted with anti-Tn-T. The cells of this axenic strain (Ax3) are often multinucleate. In interphase cells (Fig. 23) the antibody reacted with cytoplasmic microtubules, which originate from distinct organizing centres (nucleus associated bodies) to radiate in all directions; during mitosis (intranuclear), the spindle pole bodies connected by slim bundles of microtubules are reactive (Figs 24, 25). At late telophase, dividing daughter cells can be observed to regain the cytoplasmic microtubule arrays (Fig. 26). Fig. 23, bar, 5 urn; Figs 24–26, bar, 6μm.

Fig. 24.

Indirect immunofluorescence micrographs of cellular slime mould D. discoideum reacted with anti-Tn-T. The cells of this axenic strain (Ax3) are often multinucleate. In interphase cells (Fig. 23) the antibody reacted with cytoplasmic microtubules, which originate from distinct organizing centres (nucleus associated bodies) to radiate in all directions; during mitosis (intranuclear), the spindle pole bodies connected by slim bundles of microtubules are reactive (Figs 24, 25). At late telophase, dividing daughter cells can be observed to regain the cytoplasmic microtubule arrays (Fig. 26). Fig. 23, bar, 5 urn; Figs 24–26, bar, 6μm.

Fig. 25.

Indirect immunofluorescence micrographs of cellular slime mould D. discoideum reacted with anti-Tn-T. The cells of this axenic strain (Ax3) are often multinucleate. In interphase cells (Fig. 23) the antibody reacted with cytoplasmic microtubules, which originate from distinct organizing centres (nucleus associated bodies) to radiate in all directions; during mitosis (intranuclear), the spindle pole bodies connected by slim bundles of microtubules are reactive (Figs 24, 25). At late telophase, dividing daughter cells can be observed to regain the cytoplasmic microtubule arrays (Fig. 26). Fig. 23, bar, 5 urn; Figs 24–26, bar, 6μm.

Fig. 25.

Indirect immunofluorescence micrographs of cellular slime mould D. discoideum reacted with anti-Tn-T. The cells of this axenic strain (Ax3) are often multinucleate. In interphase cells (Fig. 23) the antibody reacted with cytoplasmic microtubules, which originate from distinct organizing centres (nucleus associated bodies) to radiate in all directions; during mitosis (intranuclear), the spindle pole bodies connected by slim bundles of microtubules are reactive (Figs 24, 25). At late telophase, dividing daughter cells can be observed to regain the cytoplasmic microtubule arrays (Fig. 26). Fig. 23, bar, 5 urn; Figs 24–26, bar, 6μm.

Fig. 26.

Indirect immunofluorescence micrographs of cellular slime mould D. discoideum reacted with anti-Tn-T. The cells of this axenic strain (Ax3) are often multinucleate. In interphase cells (Fig. 23) the antibody reacted with cytoplasmic microtubules, which originate from distinct organizing centres (nucleus associated bodies) to radiate in all directions; during mitosis (intranuclear), the spindle pole bodies connected by slim bundles of microtubules are reactive (Figs 24, 25). At late telophase, dividing daughter cells can be observed to regain the cytoplasmic microtubule arrays (Fig. 26). Fig. 23, bar, 5 urn; Figs 24–26, bar, 6μm.

Fig. 26.

Indirect immunofluorescence micrographs of cellular slime mould D. discoideum reacted with anti-Tn-T. The cells of this axenic strain (Ax3) are often multinucleate. In interphase cells (Fig. 23) the antibody reacted with cytoplasmic microtubules, which originate from distinct organizing centres (nucleus associated bodies) to radiate in all directions; during mitosis (intranuclear), the spindle pole bodies connected by slim bundles of microtubules are reactive (Figs 24, 25). At late telophase, dividing daughter cells can be observed to regain the cytoplasmic microtubule arrays (Fig. 26). Fig. 23, bar, 5 urn; Figs 24–26, bar, 6μm.

Anti-troponin T crossreactive components that are not microtubule-associated

Examination of the non-cellular slime mould Ph. polycephalum yielded some unexpected results. The coenocytic plasmodia of this species undergo closed mitosis. In mitotic plasmodia the anti-troponin T stained the intranuclear regions (Figs 27, 28). Comparison with specimens reacted with anti-tubulin (Figs 29, 30) showed that both antibodies labelled the polar regions of the spindle but that the patterns were not coincident, suggesting that at least some of the anti-troponin T reactive species were not located on the microtubules. Interphase plasmodia are known to lack microtubules (Havercroft & Gull, 1983). However, the anti-troponin T showed intense staining of the nuclear regions of the cell (Figs 31, 32). These discrete fluorescent spots were coincident with the location of nucleoli as seen in the phasecontrast micrograph (Fig. 32). The fact that this reaction was specific for the antibody is demonstrated by our control slide (Figs 33, 34).

Fig. 27.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 27.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 28.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 28.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 29.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 29.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 30.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 30.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 31.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 31.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 32.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 32.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 33.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 33.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 34.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Fig. 34.

Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) and corresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mould plasmodia, Ph. polycephalum, reacted with anti-Tn-T and anti-tubulin (Figs 29, 30). Mitotic plasmodia (Figs 27, 28) showed crossreaction in the regions of the mitotic apparatus as seen in corresponding phase-contrast micrograph. That the reaction is not coincident with the mitotic spindle itself is suggested by comparison with anti-tubulin staining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of the nucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, as indicated by the dark regions within nuclei in the phase-contrast micrograph (Fig. 32). The control slide incubated without any primary antibody was negative (Figs 33, 34), indicating that the reaction with the nucleoli in Fig. 34 is not due to non-specific binding to the nuclei. Bar, 12 μm.

Our examination of Pa. tetraurelia (Figs 3540) revealed some novel information. The anti-troponin T recognized what seemed to be triangular spicules at the surface of the cell (Fig. 35). These were determined to be trichocysts by indirect immunofluorescence of a fairly homogeneous population of isolated trichocysts (gift from Andrew Levin in the laboratory of Dr David Nelson, Department of Biochemistry, University of Wisconsin). The crossreactivity was localized to the tip of the trichocysts (Figs 3740). In addition, cortex preparations of paramecia revealed a regularly spaced arrangement of reactive dots suggesting the basal body distribution (Fig. 36).

Fig. 35.

Indirect immunofluorescence micrographs (Figs 35, 36, 38, 40) and corresponding phase-contrast micrographs (Figs 37, 39) of Pa. aurelia treated with anti-Tn-T. In the whole deciliated cell, triangular spicules of fluorescence were distributed randomly at the cell surface (Fig. 35). Subsequent experiments with isolated trichocysts (Figs 37-40) indicated a crossreaction with the pointed tips of these structures, which could account for the pattern seen in Fig. 35. Reactions of isolated paramecia cortices with anti-Tn-T gives rise to a well-ordered pattern of fluorescent specks of uniform distribution. These are believed to be due to crossreaction with basal bodies (Fig. 36). Fig. 35, bar, 17μm; Figs 36–40, bar, 7μm.

Fig. 35.

Indirect immunofluorescence micrographs (Figs 35, 36, 38, 40) and corresponding phase-contrast micrographs (Figs 37, 39) of Pa. aurelia treated with anti-Tn-T. In the whole deciliated cell, triangular spicules of fluorescence were distributed randomly at the cell surface (Fig. 35). Subsequent experiments with isolated trichocysts (Figs 37-40) indicated a crossreaction with the pointed tips of these structures, which could account for the pattern seen in Fig. 35. Reactions of isolated paramecia cortices with anti-Tn-T gives rise to a well-ordered pattern of fluorescent specks of uniform distribution. These are believed to be due to crossreaction with basal bodies (Fig. 36). Fig. 35, bar, 17μm; Figs 36–40, bar, 7μm.

Fig. 36.

Indirect immunofluorescence micrographs (Figs 35, 36, 38, 40) and corresponding phase-contrast micrographs (Figs 37, 39) of Pa. aurelia treated with anti-Tn-T. In the whole deciliated cell, triangular spicules of fluorescence were distributed randomly at the cell surface (Fig. 35). Subsequent experiments with isolated trichocysts (Figs 37-40) indicated a crossreaction with the pointed tips of these structures, which could account for the pattern seen in Fig. 35. Reactions of isolated paramecia cortices with anti-Tn-T gives rise to a well-ordered pattern of fluorescent specks of uniform distribution. These are believed to be due to crossreaction with basal bodies (Fig. 36). Fig. 35, bar, 17μm; Figs 36–40, bar, 7μm.

Fig. 36.

Indirect immunofluorescence micrographs (Figs 35, 36, 38, 40) and corresponding phase-contrast micrographs (Figs 37, 39) of Pa. aurelia treated with anti-Tn-T. In the whole deciliated cell, triangular spicules of fluorescence were distributed randomly at the cell surface (Fig. 35). Subsequent experiments with isolated trichocysts (Figs 37-40) indicated a crossreaction with the pointed tips of these structures, which could account for the pattern seen in Fig. 35. Reactions of isolated paramecia cortices with anti-Tn-T gives rise to a well-ordered pattern of fluorescent specks of uniform distribution. These are believed to be due to crossreaction with basal bodies (Fig. 36). Fig. 35, bar, 17μm; Figs 36–40, bar, 7μm.

Fig. 37.

Indirect immunofluorescence micrographs (Figs 35, 36, 38, 40) and corresponding phase-contrast micrographs (Figs 37, 39) of Pa. aurelia treated with anti-Tn-T. In the whole deciliated cell, triangular spicules of fluorescence were distributed randomly at the cell surface (Fig. 35). Subsequent experiments with isolated trichocysts (Figs 37-40) indicated a crossreaction with the pointed tips of these structures, which could account for the pattern seen in Fig. 35. Reactions of isolated paramecia cortices with anti-Tn-T gives rise to a well-ordered pattern of fluorescent specks of uniform distribution. These are believed to be due to crossreaction with basal bodies (Fig. 36). Fig. 35, bar, 17μm; Figs 36–40, bar, 7μm.

Fig. 37.

Indirect immunofluorescence micrographs (Figs 35, 36, 38, 40) and corresponding phase-contrast micrographs (Figs 37, 39) of Pa. aurelia treated with anti-Tn-T. In the whole deciliated cell, triangular spicules of fluorescence were distributed randomly at the cell surface (Fig. 35). Subsequent experiments with isolated trichocysts (Figs 37-40) indicated a crossreaction with the pointed tips of these structures, which could account for the pattern seen in Fig. 35. Reactions of isolated paramecia cortices with anti-Tn-T gives rise to a well-ordered pattern of fluorescent specks of uniform distribution. These are believed to be due to crossreaction with basal bodies (Fig. 36). Fig. 35, bar, 17μm; Figs 36–40, bar, 7μm.

Fig. 38.

Indirect immunofluorescence micrographs (Figs 35, 36, 38, 40) and corresponding phase-contrast micrographs (Figs 37, 39) of Pa. aurelia treated with anti-Tn-T. In the whole deciliated cell, triangular spicules of fluorescence were distributed randomly at the cell surface (Fig. 35). Subsequent experiments with isolated trichocysts (Figs 37-40) indicated a crossreaction with the pointed tips of these structures, which could account for the pattern seen in Fig. 35. Reactions of isolated paramecia cortices with anti-Tn-T gives rise to a well-ordered pattern of fluorescent specks of uniform distribution. These are believed to be due to crossreaction with basal bodies (Fig. 36). Fig. 35, bar, 17μm; Figs 36–40, bar, 7μm.

Fig. 38.

Indirect immunofluorescence micrographs (Figs 35, 36, 38, 40) and corresponding phase-contrast micrographs (Figs 37, 39) of Pa. aurelia treated with anti-Tn-T. In the whole deciliated cell, triangular spicules of fluorescence were distributed randomly at the cell surface (Fig. 35). Subsequent experiments with isolated trichocysts (Figs 37-40) indicated a crossreaction with the pointed tips of these structures, which could account for the pattern seen in Fig. 35. Reactions of isolated paramecia cortices with anti-Tn-T gives rise to a well-ordered pattern of fluorescent specks of uniform distribution. These are believed to be due to crossreaction with basal bodies (Fig. 36). Fig. 35, bar, 17μm; Figs 36–40, bar, 7μm.

Fig. 39.

Indirect immunofluorescence micrographs (Figs 35, 36, 38, 40) and corresponding phase-contrast micrographs (Figs 37, 39) of Pa. aurelia treated with anti-Tn-T. In the whole deciliated cell, triangular spicules of fluorescence were distributed randomly at the cell surface (Fig. 35). Subsequent experiments with isolated trichocysts (Figs 37-40) indicated a crossreaction with the pointed tips of these structures, which could account for the pattern seen in Fig. 35. Reactions of isolated paramecia cortices with anti-Tn-T gives rise to a well-ordered pattern of fluorescent specks of uniform distribution. These are believed to be due to crossreaction with basal bodies (Fig. 36). Fig. 35, bar, 17μm; Figs 36–40, bar, 7μm.

Fig. 39.

Indirect immunofluorescence micrographs (Figs 35, 36, 38, 40) and corresponding phase-contrast micrographs (Figs 37, 39) of Pa. aurelia treated with anti-Tn-T. In the whole deciliated cell, triangular spicules of fluorescence were distributed randomly at the cell surface (Fig. 35). Subsequent experiments with isolated trichocysts (Figs 37-40) indicated a crossreaction with the pointed tips of these structures, which could account for the pattern seen in Fig. 35. Reactions of isolated paramecia cortices with anti-Tn-T gives rise to a well-ordered pattern of fluorescent specks of uniform distribution. These are believed to be due to crossreaction with basal bodies (Fig. 36). Fig. 35, bar, 17μm; Figs 36–40, bar, 7μm.

Fig. 40.

Indirect immunofluorescence micrographs (Figs 35, 36, 38, 40) and corresponding phase-contrast micrographs (Figs 37, 39) of Pa. aurelia treated with anti-Tn-T. In the whole deciliated cell, triangular spicules of fluorescence were distributed randomly at the cell surface (Fig. 35). Subsequent experiments with isolated trichocysts (Figs 37-40) indicated a crossreaction with the pointed tips of these structures, which could account for the pattern seen in Fig. 35. Reactions of isolated paramecia cortices with anti-Tn-T gives rise to a well-ordered pattern of fluorescent specks of uniform distribution. These are believed to be due to crossreaction with basal bodies (Fig. 36). Fig. 35, bar, 17μm; Figs 36–40, bar, 7μm.

Fig. 40.

Indirect immunofluorescence micrographs (Figs 35, 36, 38, 40) and corresponding phase-contrast micrographs (Figs 37, 39) of Pa. aurelia treated with anti-Tn-T. In the whole deciliated cell, triangular spicules of fluorescence were distributed randomly at the cell surface (Fig. 35). Subsequent experiments with isolated trichocysts (Figs 37-40) indicated a crossreaction with the pointed tips of these structures, which could account for the pattern seen in Fig. 35. Reactions of isolated paramecia cortices with anti-Tn-T gives rise to a well-ordered pattern of fluorescent specks of uniform distribution. These are believed to be due to crossreaction with basal bodies (Fig. 36). Fig. 35, bar, 17μm; Figs 36–40, bar, 7μm.

Another example of anti-troponin T crossreactivity in the vicinity of the mitotic apparatus was seen in mitotic sea-urchin eggs (5. purpuratus) (Figs 4143). The staining pattern here could be described as centrospheral, with lack of reactivity in the spindle region of the mitotic apparatus. The distribution was similar to those obtained by Kuriyama & Borisy (1985) with monoclonal antibodies raised against isolated sea-urchin mitotic apparatus. Anti-tubulin staining of sea-urchin eggs processed at the same time indicated the characteristic array of microtubules (Fig-43).

Fig. 41.

Indirect immunofluorescence of sea-urchin eggs (S. purpuratus). Anti-Tn-T showed intense regions of fluorescence at pericentrosomal regions of the mitotic apparatus. A clear region (non-reactive) is seen at the centrosome (Figs 41, 42). Samples of eggs from the same batch were also processed with anti-tubulin; the characteristic microtubule arrays were observed (Fig. 43). Fig. 41, bar, 19μm; Figs4243, bar, 28 μm.

Fig. 41.

Indirect immunofluorescence of sea-urchin eggs (S. purpuratus). Anti-Tn-T showed intense regions of fluorescence at pericentrosomal regions of the mitotic apparatus. A clear region (non-reactive) is seen at the centrosome (Figs 41, 42). Samples of eggs from the same batch were also processed with anti-tubulin; the characteristic microtubule arrays were observed (Fig. 43). Fig. 41, bar, 19μm; Figs4243, bar, 28 μm.

Fig. 42.

Indirect immunofluorescence of sea-urchin eggs (S. purpuratus). Anti-Tn-T showed intense regions of fluorescence at pericentrosomal regions of the mitotic apparatus. A clear region (non-reactive) is seen at the centrosome (Figs 41, 42). Samples of eggs from the same batch were also processed with anti-tubulin; the characteristic microtubule arrays were observed (Fig. 43). Fig. 41, bar, 19μm; Figs42–43, bar, 28 μm.

Fig. 42.

Indirect immunofluorescence of sea-urchin eggs (S. purpuratus). Anti-Tn-T showed intense regions of fluorescence at pericentrosomal regions of the mitotic apparatus. A clear region (non-reactive) is seen at the centrosome (Figs 41, 42). Samples of eggs from the same batch were also processed with anti-tubulin; the characteristic microtubule arrays were observed (Fig. 43). Fig. 41, bar, 19μm; Figs42–43, bar, 28 μm.

Fig. 43.

Indirect immunofluorescence of sea-urchin eggs (S. purpuratus). Anti-Tn-T showed intense regions of fluorescence at pericentrosomal regions of the mitotic apparatus. A clear region (non-reactive) is seen at the centrosome (Figs 41, 42). Samples of eggs from the same batch were also processed with anti-tubulin; the characteristic microtubule arrays were observed (Fig. 43). Fig. 41, bar, 19μm; Figs42–43, bar, 28 μm.

Fig. 43.

Indirect immunofluorescence of sea-urchin eggs (S. purpuratus). Anti-Tn-T showed intense regions of fluorescence at pericentrosomal regions of the mitotic apparatus. A clear region (non-reactive) is seen at the centrosome (Figs 41, 42). Samples of eggs from the same batch were also processed with anti-tubulin; the characteristic microtubule arrays were observed (Fig. 43). Fig. 41, bar, 19μm; Figs42–43, bar, 28 μm.

We have used a monoclonal antibody raised against skeletal troponin T to localize anti-troponin T crossreactive components in a variety of non-muscle cells. While it is not clear whether the epitopes recognized by the antibody are from related or different molecules, the specificity of a monoclonal antibody for a single determinant does imply that some real structural similarity must exist between the recognized determinants. Several instances of crossreacting sites on different molecules have been detected by the recent use of monoclonal antibodies (Lin et al. 1984). The biological significance of some are difficult to explain (Blose et al. 1982; Dulbecco et al. 1981), but others suggest that monoclonal antibodies can detect functional sites common to different molecules (Lane & Hoeffler, 1980; Lane & Kaprowski, 1982; Wang et al. 1983).

In addition to strong crossreaction with a 35×103Mr protein in HeLa cells, some crossreaction was also observed between the anti-troponin T and a 55×103Afr protein, presumably HeLa tubulin. Such crossreactivity would not be unexpected, given the sequence homology between the N terminus of troponin T and the C terminus of porcine brain tubulin (Krauhs et al. 1981). However, no reaction with tubulin was observed upon incubation of the anti-troponin T with blots of porcine brain microtubule protein (Fig. 5, lane 5). Instead, strong crossreactivity was observed with high molecular weight polypeptides associated with microtubules (HMW MAPs). A similar crossreaction was observed with immunoblots of cerebellar homogenate (Fig. 18B, lane 2). While it is possible that crossreaction with betatubulin could account for the microtubule pattern seen in indirect immunofluorescence, we do not think that is the case, for the following reasons: (1) the crossreaction with beta-tubulin on immunoblots was seen only at high concentrations of antibody, suggesting a low-affinity crossreaction; (2) the punctate nature of the anti-troponin T staining pattern was markedly different from the anti-tubulin staining (compare Figs 3 and 4); (3) immunoblots of whole cell and cytoskeletal homogenates indicated that the 35 ×103Mr crossreactive protein was retained in the taxol-stabilized cytoskeletons after lysis. Since no staining was observed with other cytoskeletal components (stress fibres, intermediate filaments), we assume that the 35×103Mr protein observed in immunoblots was the microtubule-associated crossreactive component observed by indirect immunofluorescence.

The structural similarity implied by crossreactivity of polypeptides with the antitroponin T could indicate some common macromolecular interaction. The function of microtubules (cell division, cell shape changes and secretion) is closely coupled to their ability to depolymerize and polymerize. Microtubule assembly-disassembly has been shown to be Ca2+-sensitive (Weisenberg, 1972), the effect of Ca2+ possibly being mediated by calmodulin, a protein that functionally parallels Tn-C from striated muscle (Marcum et al. 1978). The characteristic morphological location of calmodulin is in the chromosome-to-pole regions of the mitotic apparatus as seen by indirect immunofluorescence microscopy and lends further credence to this idea (Welsh et al. 1971, 1979).

Unlike Tn-C, whose calcium binding effect is mediated through binding to Tn-I and Tn-T, the binding mechanism of the Ca2+-calmodulin complex to microtubules is poorly understood. Some evidence points to the binding of calmodulin to the high molecular weight microtubule-associated protein, MAP 2 (Lee & Wolff, 1982; Rebhun et al. 1980) as well as to the lower molecular weight (55–62(×103)Mr microtubule-associated proteins collectively known as tau proteins (Weingarten et al. 1975). On the basis of our morphological results, we propose the following analogy: in muscle, Tn-I and Tn-T mediate the action of Ca2+(via Tn-C) on thin filaments; in non-muscle cells, a molecule homologous to troponin-T mediates the action of Ca2+(via calmodulin) on microtubules.

Crossreactive components not associated with microtubules have also been observed in our survey. Examination of these instances also revealed that the distribution of the troponin T crossreactive species is in regions in which calcium or calmodulin activity had been described. In our observations of fertilized sea-urchin eggs, the distribution of anti-troponin T around the pericentrosomal regions of whole egg mitotic apparatus is similar to that observed when cells are labelled with 7-chlorotetracycline, a reagent that forms a fluorescent chelate with divalent cations in a hydrophobic environment (Wolniaket al. 1983).

The morphological distribution of anti-troponin T in Pa. aurelia also approximates that previously described for related Ca2+–calmodulin activity in these organisms. The localization of the crossreactive component in trichocysts is consistent with what little is known about these organelles. These membrane-bound organelles are docked beneath the plasma membrane at specific secretory sites in the highly organized cell cortex (Hidaka et al. 1979). The trichocysts remain docked in the secretory sites until a stimulus triggers release of the vesicle content (trichocyst matrix) via exocytosis. The trichocyst matrix has been recently found to contain significant amounts of the Ca2+-regulatory protein, calmodulin (Rauh & Nelson, 1981). The role of the matrix calmodulin, which appears to be a structural part of the trichocyst, is not understood. However, trifluoperazine, a drug known to inhibit calmodulin-regulated enzymes, inhibits trichocyst release in Paramecium (Garofalo et al. 1983). This result suggests the possible involvement of calmodulin at some stage of the release process in these cells. In addition, anti-troponin T revealed crossreaction in isolated cortex preparations, suggesting basal body distribution. Again, calmodulin has been observed in the basal bodies of cilia from Paramecium, Tetrahymena and the fresh-water mussel Elliptio (Means & Dedman, 1980).

In summary, we have presented evidence that anti-troponin T crossreactive components are widespread throughout the animal, plant and fungal kingdoms. The relatedness of the crossreacting proteins to troponin T in terms of structure and function cannot be adequately resolved. However, the extreme evolutionary persistence of the microtubule-associated epitope suggests conservation of a particular site due to some fundamental requirement in cellular function. The localization of other crossreactive components to regions of Caz+-related activity further suggests some common function between microtubule-associated and non-microtubule-associated epitopes, possibly relating to Ca2+ regulation. Further progress in this field will require isolation of the crossreactive species and its molecular analysis.

We are grateful to the following for gifts of cells: Drs Philippa Claude, Randall Dimond, Ryoko Kuriyama, Andrew Levin, Eileen Paul and Dale Vandre. We thank Dr John Fallon and Ms Rebecca Fuldner for use of equipment and their expertise in paraffin-section immunocytochemistry. We also thank Ms Carmen Huston and Ms Sandy Damrauer for typing the manuscript. This investigation was supported by a National Science Foundation grant (PCM 8309286) to S.S.L. and a National Institutes of Health grant (GM 30385) to G.G.B.

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