J. CellSci. 85, 1-19(1986)Printed in Great Britain © The Company of Biologists Limited 1986

WIDESPREAD OCCURRENCE OF ANTI-TROPONIN T

CROSSREACTIVE COMPONENTS IN NON-MUSCLE

CELLS

SOO-SIANG L I M \ GORDON E. HERING2 AND GARY G. BORISY1 3

1Laboratory of Molecular Biology, 2Department of Pathology and ^Department of Zoology,University of Wisconsin, Madison, Wisconsin 53706, USA

SUMMARY

Using a monoclonal antibody generated against striated muscle troponin T, we previously notedthe presence of crossreactive components in smooth muscle and non-muscle cells. Since thepresence of troponin T in tissues other than striated muscle is controversial, we sought to establishthe nature of the crossreaction and to determine the extent of its occurrence. For this study,indirect immunofluorescence microscopy and immunoblot analyses were performed. Crossreactivematerial was found in diverse cells from the animal, plant and fungal kingdoms. On the basis ofmorphological distributions, both microtubule-associated and non-microtubule-associated com-ponents were revealed. Microtubule-associated components of animal cell lines included a35xlO3Mr protein, similar in electrophoretic mobility to skeletal troponin T (37xlO3Mr).Reactive components of comparable mobility were observed in immunoblots of brain andcerebellar homogenates. Filamentous staining was observed in a variety of mammalian cells inculture and in cells of vertebrate tissues. Chick cerebellar tissue showed reactions in the neurites ofthe molecular layer and granule cell bodies. In the plant kingdom, examination of the onion root-tipcells 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. Non-microtubule-associated components were observed in the centrosphere regions of mitotic sea-urchin eggs, in mitotic and interphase plasmodia of Physarum polycephalum, and in trichocystsand basal bodies of Paramecium tetraurelia. In all systems examined, the troponin T crossreactivecomponents were located in regions or on structures of possible Ca2+ or calmodulin activity,suggesting a possible functional similarity to troponin T.

INTRODUCTION

The calcium control of striated muscle contraction is regulated through troponin-tropomyosin interactions. Troponin is a complex of three subunits designated Tn-I,Tn-C and Tn-T (Greaser & Gergely, 1971). Tn-C is the calcium binding componentthat interacts with Tn-I to relieve the inhibition of the latter on the ATPase activityof actomyosin. However, it is only in the presence of all three troponin subunits thatthe system becomes sensitive to calcium ions. In this respect Tn-T, which has a highaffinity 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).

Key words: anti-troponin T, monoclonal, non-muscle.

2 S.-S. Lim, G. E. Hering and G. G. Borisy

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

Previously, we reported the production of a monoclonal antibody generatedagainst striated muscle troponin T that crossreacted with troponin T in all muscletypes (Lim et al. 1984) and that crossreacted with components in smooth muscle andnon-muscle cells (Lim et al. 1985). In this study, we report the immunochemicalcrossreaction of this anti-troponin T with components in animal, plant and fungalkingdoms. Its widespread occurrence in cells of diverse phylogenetic origin impliesthe conservation of a biologically important determinant. On the basis of morpho-logical distribution as determined by indirect immunofluorescence microscopy, twogroups of anti-troponin T crossreactive components were observed: those associatedwith microtubules and those not associated with microtubules. The localization ofthe crossreactive components in regions or structures of possible Ca2+ or calmodulinactivity suggests that an epitope shared with troponin T may be of general regulatorysignificance in non-muscle cells.

MATERIALS AND METHODS

Monoclonal antibodiesThe 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 ratmonoclonal antibody (IgG) against tubulin was a gift from Dr J. Kilmartin (Kilmartineia/. 1982).

Indirect immunofluorescence microscopyCell lines. Mouse 3T3 fibroblasts, 356 human foreskin fibroblasts, rat kangaroo kidney (PtKi)

cells, neuroblastoma (N2A) cells and Chinese hamster ovary (CHO) cells were grown on glasscoverslips in Ham's F-10 medium supplemented with 10% foetal bovine serum (FBS). HeLa cellswere grown in F-10 supplemented with 10% horse-serum. Cells plated on coverslips were rinsedtwice 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 threewashes in PBS, the cells were treated with lmgml" 1 sodium borohydride for l h . After threewashes in PBS, the cells were incubated for l h with the appropriate primary antibody (45%(NH4)2SO4-cut hybridoma supernatant, 2-3 mgml"1) at 37°C. This was followed by three washesin 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 thenmounted in a medium containing lmgml""1 />-phenylenediamine as an anti-bleaching agent(Sammak & Borisy, unpublished data).

For double-label indirect immunofluorescence microscopy incubation of each primary antibodywas followed by its corresponding secondary antibody. For example, anti-troponin T was followed

Anti-troponin T in non-muscle cells 3

by affinity-purified goat anti-mouse IgM (^-chain-specific, FITC-conjugated; Cappel Labs), afterwhich the second primary antibody, anti-tubulin, was followed by goat anti-rat IgG (gamma-chain-specific, RITC-conjugated; Cappel Labs). Cells were examined with a Zeiss Universal epi-fluorescence microscope equipped with narrow band fluorescein and rhodamine excitation filters.

In control experiments, primary antibody from hybridomas was substituted by PBS or cellsupernatants from hybridoma secreting antibodies to unrelated antigens (gift from Dr PatriciaWitt, Molecular Biology Laboratory, University of Wisconsin). In double-label indirect immuno-fluorescence control experiments, the goat anti-mouse IgM (^-chain-specific) FITC was reactedwith 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 in95% ethanol/5% ethanol/5% acetic acid and prepared for paraffin embedment and indirectimmunofluorescence microscopy according to the procedure of Saint-Marie (1962). Sections ofabout 4 p n were cut and stained.

Plant cell preparation. Seeds of onion (Allium) were germinated in the dark at room temperatureon moist filter paper. When the roots were about 1 cm long, the apical 1-4 mm was cut and fixed for1 h in freshly prepared 3-7% formaldehyde in PBS, pH7-4, containing 50mM-EGTA. Aftertreatment with 1% cellulysin (Sigma) in 0-4M-mannitol and SmM-EGTA (Wicked al. 1981), theroot tips were again rinsed in buffer and squashed between coverslips to release individual cells.

Dictyostelium discoideum. An axenic strain of the cellular slime mould (Ax3) was a gift from thelaboratory of Dr Randall Dimond (Department of Bacteriology, University of Wisconsin). Afterthe density of the cells was determined under the phase-contrast microscope, the cells were allowedto settle onto polylysine-coated coverslips for about 30min, then fixed with 3-7% formaldehyde inPBS, pH 7-4. After permeabilization with both methanol and acetone, the cells were processed forimmunofluorescence microscopy as described by White et al. (1983).

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

Sea-urchin (Strongylocentrotus purpuratus) eggs. Sea-urchin egg samples were a gift from DrRyoko Kuriyama (Department of Anatomy, University of Minnesota). Fertilized eggs wereallowed to develop through first mitosis. After checking the density of the eggs under the phase-contrast microscope, they were allowed to settle onto polylysine-coated coverslips and then fixed in2% glutaraldehyde in 0-4M-sodium acetate buffer, pH 6-0, with 1% Triton X-100 and SOmM-EGTA (Asnes & Schroeder, 1979). The coverslips were then routinely processed for indirectimmunofluorescence microscopy.

Paramecium tetraurelia. Samples of Paramecia were provided by Andrew Levin from thelaboratory of Dr David Nelson (Department of Biochemistry, University of Wisconsin). The cellswere deciliated in 100 mM-MnCl2 and fixed with 3-7 % paraformaldehyde in 60 mM-PIPES, 25 mM-HEPES, 10 mM-EGTA and 2 mM-MgCl2 (PHEM), pH 6-9. After they were allowed to settle ontopolylysine-coated coverslips, the cells were processed as usual for indirect immunofluorescencemicroscopy.

SDS-polyacrylamide gel electrophoresis and immunoblottingPreparation 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 withlOjUgml"1 taxol for 15min, followed by treatment (90s) with lysis buffer containing 10mM-PIPES, pH6-9, 0-2%NP-40, 5 ^gml" ' taxol and 1 mM-phenylmethylsulphonyl fluoride (PMSF).After solubilization in hot sample buffer all samples were further lysed by three 30-s pulses from aHeat Systems Sonifier (Branson Ultrasonics, Plainview, NY), setting 3. Samples were then appliedto polyacrylamide gels (Laemmli, 1970). Preparations of myofibrils (Wang, 1982) and chickcerebellum were homogenized at 0°C in 0-1 M-Tris-HCl buffer, pH7-3, containing 1-OmM-PMSF. Samples were then resuspended in hot sample buffer and boiled for 15 min.

After electrophoretic separation, the polypeptides were transferred electrophoretically ontonitrocellulose paper (Towbin et al. 1979). The blots were stained for protein in 0-1% AmidoBlack in 45% methanol and 10% acetic acid, while duplicate blots were treated with a

4 S.-S. Lim, G. E. Hering and G. G. Borisy

blocking solution (20mM-Tris, 0-9% NaCl, 10% horse serum) and then reacted with a 1:1000dilution of anti-troponin T ascitic fluid (5-6 mg ml~' IgM). To visualize specific antigen-antibodyreactions, the enzyme immunoassay kit and protocol from BIO-RAD laboratories was used (BIO-RAD Immuno-blot AGR-HRP Assay Kit). Controls for the immunoblot experiments includedsubstitution of the blocking buffer for primary antibody and non-immune mouse IgM.

RESULTS

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

Mammalian cells. Several mammalian cell lines were examined, including 3T3mouse fibroblasts, 356 human foreskin fibroblasts, PtKi cells, CHO cells and HeLacells. Both interphase and mitotic cells reacted with the antibody as revealed byindirect immunofluorescence microscopy in all cell lines. In interphase cells (Fig. 1)brightly stained punctate networks of cytoplasmic filaments were observed. Thesefilaments converged on a perinuclear focus. The anti-troponin T (Anti-Tn-T) alsoidentified crossreactive components on midbodies and mitotic spindles of dividingcells (Fig. 2). These morphological patterns suggested microtubule distribution. Toverify this, double-label indirect immunofluorescence microscopy was performed,which permitted the simultaneous visualization of two antigens in the same cell. Thefilamentous pattern recognized by the anti-troponin T was spatially coincident withthat of microtubules, as indicated by anti-tubulin. Higher-magnification micro-graphs (Figs 3, 4) also show very clearly the punctate nature of the anti-troponin Tstaining pattern at this dilution (Fig. 3) as compared to the linear pattern of theanti-tubulin staining (Fig. 4). The fact that the anti-troponin T decorated onlymicrotubules and no other cytoskeletal elements (e.g. stress fibres, intermediatefilaments) was also apparent.

To obtain information concerning the molecular weight of the anti-troponin Tcrossreactive species, immunoblots containing samples of HeLa whole cell hom-ogenate (Fig. 5, lane 3) and HeLa cytoskeletons (Fig. 5, lane 4) were treated withanti-troponin T. (Compare with the reactive troponin T band in the myofibrilpreparation, lane 2.) A strong reaction with a 35 X 103Mr band was detected in bothHeLa cell samples. The retention of the reactive 35xl03Mr band in the cellcytoskeleton fraction (Fig. 5, lane 4) confirmed our indirect immunofluorescenceobservations that the anti-troponin T crossreactive species was associated with acomponent of the cytoskeleton, most probably microtubules. A faint reaction wassometimes observed at the 55x10 Mr band of both HeLa samples. Immunoblots oftwo-dimensional gels (data not shown) indicated this to be due to a crossreaction withbeta-tubulin (see Discussion). However, no reaction was observed with the 55X103Mr tubulin band from the brain microtubule protein preparation in lane 5, eventhough strong reactions were apparent with some high molecular weight poly-peptides (see Discussion).

Nerve cells and tissues. Paraffin sections through the thoracic regions of 5-daychick embryos revealed crossreaction with a major non-muscle tissue mass. In

Anti-troponin T in non-muscle cells S.

addition to the reaction with the somitie regions (skeletal muscle precursor) andforming myocardium, cellular components of the neural tube as well as fibre tractswere intensely stained (Figs 6, 7). Since the resolution of tissue sections (particularly4-jUm paraffin sections) was inadequate to determine whether the crossreactivecomponent observed in nerve cells was microtubule-associated, we decided to ex-amine 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 ganglioncells from rat (gift from Dr Philippa Claude, Primate Center, University of

Fig. 1. Interphase fibroblasts (356 human foreskin fibroblast) reacted with the anti-Tn-T reveal staining of a cytoplasmic filamentous array with a perinuclear focus. Bar, 10 £ttn.

Fig. 2. Mitotic fibroblasts (356 human foreskin fibroblast) reacted with anti-Tn-Tindicate reaction with the mitotic apparatus. Bar, 10 fim.

Figs 3, 4. Double-label indirect immunofluorescence (356 human foreskin fibroblast)with anti-Tn-T followed by FITC-conjugated anti-mouse IgM and anti-tubulin followedby 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, 8jUm.

6 5.-5. Urn, G. E. Hering and G. G. Borisy

Wisconsin) also revealed filamentous staining in the neurites (Figs 10, 11). Intensestaining was also observed in the cell bodies. However, as these regions are quitethick, it was not possible to observe any detail in the staining pattern.

Mrx10"3

B

92-5-

66-2-

1 2 3 1 2 3 2 3

Fig. 5. Immunoblot of HeLa cell samples and porcine brain microtubule protein, bothreacted with the anti-Tn-T. A. Coomassie Brilliant Blue-stained SDS-5% to 15%polyacrylamide gradient gel; B, 0-1% Amido Black-stained nitrocellulose containingtransferred proteins; C, nitrocellulose containing transferred proteins stained with1:1000 anti-Tn-T ascites fluid followed by 1:1000 peroxidase-conjugated goat anti-mouse IgM (^-chain-specific), colour development with 4-chloro-l-naphthol. Lane 1,molecular weight standards; lane 2, bovine cardiac myofibrils; lane 3, HeLa cellhomogenate; lane 4, HeLa cytoskeletons; lane 5, twice-cycled porcine brain microtubuleprotein. Note the similar mobility of troponin T (C, lane 2) and the HeLa cross-reactive species (C, lanes 3,4). Despite sufficient transfer of brain tubulin for detection(C, lane 5), no reaction was observed with the 55 K protein, but strong reactions wereobserved with some high molecular weight peptides. No high molecular weight reactivecomponent is seen in HeLa cells or cytoskeletons (C, lanes 3, 4).

Anti-troponin T in non-muscle cells

Figs 6, 7. Indirect immunofluorescence using anti-Tn-T on paraffin sections throughthoracic region of 5-day chick embryo (transverse sections). Fig. 6 shows that in additionto reaction with the somitic regions (s), intense staining is seen at the neural tube (w) aswell as the forming spinal nerve (sn). Higher magnification (Fig. 7) shows reaction withfibre tract at floor of neural tube. Fig. 6, bar, 100 fim; Fig. 7, bar, 2/im.

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

Figs 10, 11. Indirect immunofluorescence on primary cultures of dissociated para-sympathetic ganglion cells from rat. anti-Tn-T staining reveals intense reaction with cellbodies (c) as well as filamentous arrays in neurite. Bar, 8 Jim.

8 S.-S. Lim, G. E. Hering and G. G. Borisy

Paraffin sections of newly hatched chick brain and cerebellum also showed anabundance of the crossreactive component. In the cerebellum (Figs 12—17) thepattern 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).

Immunoblots of brain microtubule protein (Fig. 5) indicated a crossreaction witha high molecular weight species identified as MAP IA (unpublished data). Whilecertain features of the anti-troponin T crossreaction were similar to that reported byBloom 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 whitematter differed from the reported MAP IA distribution. This suggested the possi-bility 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 inbrain, two reactive bands were observed in the cerebellar homogenate (Fig. 18):bands corresponding to MAP I and a lower molecular weight band at about35(X 103)Mr. This lower molecular weight band was not present in our immunoblotsof 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.

Plant cells. Examination of meristematic cells of onion {Allium) root revealed apattern of anti-troponin T crossreaction very similar to that described by others formicrotubule distribution. In interphase cells (Fig. 19), cortical arrays of well-alignedmicrotubules were observed while the nuclear region was non-reactive. In cells thatwere 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 mitoticcells (Fig. 21) the anti-troponin T reacted with the spindle fibres as well as the polarcap regions onto which the microtubules converge. At late anaphase anti-troponin Tstaining of the phragmoplast region was evident. The co-localization of the anti-troponin T crossreactive components with microtubules of the onio.i cells wasfurther confirmed by double-label indirect immunofluorescence (data not shown).

Dictyostelium discoideum. The organization of the microtubules in the cellularslime mould has been previously visualized using antibodies against tubulin (Cappu-ccinelli et al. 1982). The anti-troponin T staining of an axenic strain (Ax3) revealed apattern similar to that reported for anti-tubulin. The cells of this strain are oftenmultinucleate. In interphase cells (Fig. 23) the antibody reacted with cytoplasmicmicrotubules that originated from distinct microtubule-organizing centres (nucleus-associated bodies). In mitotic cells (Figs 24, 25) the interphase network of micro-tubules was replaced by the intranuclear pole body. In early prophase the antibodyreacted with the spindle pole bodies, which had duplicated and were now connectedby short bundles of microtubules (Fig. 24). These increased in length as mitosisprogressed (Fig. 25). At telophase cytoplasmic microtubules in the dividingdaughter cells were again apparent (Fig. 26). In all instances the co-localization of

Anti-troponin Tin non-muscle cells

Figs 12-17. Indirect immunofluorescent micrographs of paraffin-embedded sections ofnewly hatched chick cerebellum. Anti-Tn-T is seen in both the molecular layer (in) andgranular layer (g). In the molecular layer, staining of neurites as well as some cell bodiesis 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 theinternal white matter except for that with some granule cells (Fig. 17). Fig. 12, bar,45pm; Fig. 13, bar, 22jitm; Figs 14-17, bar, 19jun.

10 5.-5. Lint, G. E. Hering and G. G. Borisy

the anti-troponin T staining with that of anti-tubulin was further confirmed bydouble-label indirect immunofluorescence in these cells (data not shown).

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

Examination of the non-cellular slime mould Ph. polycephalum yielded some un-expected results. The coenocytic plasmodia of this species undergo closed mitosis. Inmitotic plasmodia the anti-troponin T stained the intranuclear regions (Figs 27, 28).Comparison with specimens reacted with anti-tubulin (Figs 29, 30) showed that bothantibodies labelled the polar regions of the spindle but that the patterns were notcoincident, suggesting that at least some of the anti-troponin T reactive species were

B\ r

i1 2 1 2

Fig. 18 Immunoblot of chick cerebellar homogenate reacted with the anti-Tn-T.A. Amido Black-stained nitrocellulose to visualize transferred proteins; B, transferredproteins reacted with anti-Tn-T; lanes 1, myofibril preparation; lanes 2, chick cerebellarhomogenate. 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.

Anti-troponin T in non-muscle cells 11

Fig. 19-22. 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), thepre-prophase band, which predicts the site and plane of future cytokinesis (Fig. 20), themitotic spindle (Fig. 21) and the phragmoplast (p) (Fig. 22). Bar, 8jum.

not located on the microtubules. Interphase plasmodia are known to lackmicrotubules (Havercroft & Gull, 1983). However, the anti-troponin T showedintense staining of the nuclear regions of the cell (Figs 31, 32). These discretefluorescent spots were coincident with the location of nucleoli as seen in the phase-contrast micrograph (Fig. 32). The fact that this reaction was specific for theantibody is demonstrated by our control slide (Figs 33, 34).

Our examination of Pa. tetraurelia (Figs 35—40) revealed some novel information.The anti-troponin T recognized what seemed to be triangular spicules at the surfaceof the cell (Fig. 35). These were determined to be trichocysts by indirect immuno-fluorescence of a fairly homogeneous population of isolated trichocysts (gift fromAndrew Levin in the laboratory of Dr David Nelson, Department of Biochemistry,University of Wisconsin). The crossreactivity was localized to the tip of thetrichocysts (Figs 37—40). In addition, cortex preparations of paramecia revealed aregularly spaced arrangement of reactive dots suggesting the basal body distribution(Fig. 36).

12 S.-S. Lint, G. E. Hering and G. G. Borisy

Another example of anti-troponin T crossreactivity in the vicinity of the mitoticapparatus was seen in mitotic sea-urchin eggs (5. purpuratus) (Figs 41-43). Thestaining pattern here could be described as centrospheral, with lack of reactivity inthe spindle region of the mitotic apparatus. The distribution was similar to thoseobtained by Kuriyama & Borisy (1985) with monoclonal antibodies raised againstisolated sea-urchin mitotic apparatus. Anti-tubulin staining of sea-urchin eggsprocessed at the same time indicated the characteristic array of microtubules(Fig. 43).

DISCUSSION

We have used a monoclonal antibody raised against skeletal troponin T to localizeanti-troponin T crossreactive components in a variety of non-muscle cells. While it is

Figs 23-26. Indirect immunofluorescence micrographs of cellular slime mould D. discoi-deum reacted with anti-Tn-T. The cells of this axenic strain (Ax3) are often multinu-cleate. In interphase cells (Fig. 23) the antibody reacted with cytoplasmic microtubules,which originate from distinct organizing centres (nucleus associated bodies) to radiate inall directions; during mitosis (intranuclear), the spindle pole bodies connected by slimbundles of microtubules are reactive (Figs 24, 25). At late telophase, dividing daughtercells can be observed to regain the cytoplasmic microtubule arrays (Fig. 26). Fig. 23, bar,S ; Figs 24-26, bar, 6

Anti-troponin Tin non-muscle cells 13

not clear whether the epitopes recognized by the antibody are from related ordifferent molecules, the specificity of a monoclonal antibody for a single determinantdoes imply that some real structural similarity must exist between the recognizeddeterminants. Several instances of crossreacting sites on different molecules havebeen detected by the recent use of monoclonal antibodies (Lin et al. 1984). Thebiological significance of some are difficult to explain (Blose et al. 1982; Dulbeccoet al. 1981), but others suggest that monoclonal antibodies can detect functional sitescommon to different molecules (Lane & Hoeffler, 1980; Lane & Kaprowski, 1982;Wang etal. 1983).

In addition to strong crossreaction with a 35X 103Afr protein in HeLa cells, somecrossreaction was also observed between the anti-troponin T and a 55Xl03A/r

protein, presumably HeLa tubulin. Such crossreactivity would not be unexpected,given the sequence homology between the N terminus of troponin T and the Cterminus of porcine brain tubulin (Krauhs et al. 1981). However, no reaction withtubulin was observed upon incubation of the anti-troponin T with blots of porcinebrain microtubule protein (Fig. 5, lane 5). Instead, strong crossreactivity wasobserved with high molecular weight polypeptides associated with microtubules(HMW MAPs). A similar crossreaction was observed with immunoblots of cerebellarhomogenate (Fig. 18B, lane 2). While it is possible that crossreaction with beta-tubulin could account for the microtubule pattern seen in indirect immunofluor-escence, we do not think that is the case, for the following reasons: (1) thecrossreaction with beta-tubulin on immunoblots was seen only at high concentrationsof antibody, suggesting a low-affinity crossreaction; (2) the punctate nature of theanti-troponin T staining pattern was markedly different from the anti-tubulinstaining (compare Figs 3 and 4); (3) immunoblots of whole cell and cytoskeletalhomogenates indicated that the 35 X 103Mr crossreactive protein was retained in thetaxol-stabilized cytoskeletons after lysis. Since no staining was observed with othercytoskeletal components (stress fibres, intermediate filaments), we assume that the35XlO3Afr protein observed in immunoblots was the microtubule-associated cross-reactive component observed by indirect immunofluorescence.

The structural similarity implied by crossreactivity of polypeptides with the anti-troponin T could indicate some common macromolecular interaction. The functionof microtubules (cell division, cell shape changes and secretion) is closely coupled totheir ability to depolymerize and polymerize. Microtubule assembly—disassemblyhas been shown to be Ca2+-sensitive (Weisenberg, 1972), the effect of Ca2+ possiblybeing mediated by calmodulin, a protein that functionally parallels Tn-C fromstriated muscle (Marcum et al. 1978). The characteristic morphological location ofcalmodulin is in the chromosome-to-pole regions of the mitotic apparatus as seen byindirect immunofluorescence microscopy and lends further credence to this idea(Welshes a/. 1971, 1979).

Unlike Tn-C, whose calcium binding effect is mediated through binding to Tn-Iand Tn-T, the binding mechanism of the Ca — calmodulin complex to microtubulesis poorly understood. Some evidence points to the binding of calmodulin to the highmolecular weight microtubule-associated protein, MAP 2 (Lee & Wolff, 1982;

14 S.-S. Lim, G. E. Hering and G. G. Borisy

Anti-troponin T in non-muscle cells 15

Rebhun et al. 1980) as well as to the lower molecular weight (55-62(XlO3)A/r

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 ofCa2+ (via calmodulin) on microtubules.

Crossreactive components not associated with microtubules have also been ob-served in our survey. Examination of these instances also revealed that the dis-tribution of the troponin T crossreactive species is in regions in which calcium orcalmodulin activity had been described. In our observations of fertilized sea-urchineggs, the distribution of anti-troponin T around the pericentrosomal regions ofwhole egg mitotic apparatus is similar to that observed when cells are labelled with7-chlorotetracycline, a reagent that forms a fluorescent chelate with divalent cationsin a hydrophobic environment (Wolniakef al. 1983).

The morphological distribution of anti-troponin T in Pa. aurelia also approxi-mates that previously described for related Ca2+—calmodulin activity in theseorganisms. The localization of the crossreactive component in trichocysts is con-sistent with what little is known about these organelles. These membrane-boundorganelles are docked beneath the plasma membrane at specific secretory sites in thehighly organized cell cortex (Hidaka et al. 1979). The trichocysts remain docked inthe secretory sites until a stimulus triggers release of the vesicle content (trichocystmatrix) via exocytosis. The trichocyst matrix has been recently found to containsignificant amounts of the Ca2+-regulatory protein, calmodulin (Rauh & Nelson,1981). The role of the matrix calmodulin, which appears to be a structural part of thetrichocyst, is not understood. However, trifluoperazine, a drug known to inhibitcalmodulin-regulated enzymes, inhibits trichocyst release in Paramecium (Garofaloet al. 1983). This result suggests the possible involvement of calmodulin at somestage of the release process in these cells. In addition, anti-troponin T revealedcrossreaction 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 crossreactivecomponents are widespread throughout the animal, plant and fungal kingdoms. Therelatedness of the crossreacting proteins to troponin T in terms of structure and

Figs 27-34. Indirect immunofluorescence micrographs (Figs 27, 29, 31, 33) andcorresponding phase-contrast micrographs (Figs 28, 30, 32, 34) of acellular slime mouldplasmodia, 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 mitoticapparatus as seen in corresponding phase-contrast micrograph. That the reaction is notcoincident with the mitotic spindle itself is suggested by comparison with anti-tubulinstaining (Figs 29, 30). In interphase plasmodia, anti-Tn-T shows intense staining of thenucleolar regions (Figs 31, 32). The nuclei of this strain often have multiple nucleoli, asindicated 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 bindingto the nuclei. Bar, 12 jUm.

37

39 -Figs 35—40. Indirect immunofluorescence micrographs (Figs 35, 36, 38, 40) andcorresponding phase-contrast micrographs (Figs 37, 39) of Pa. aurelia treated with anti-Tn-T. In the whole deciliated cell, triangular spicules of fluorescence were distributedrandomly at the cell surface (Fig. 35). Subsequent experiments with isolated trichocysts(Figs 37-40) indicated a crossreaction with the pointed tips of these structures, whichcould account for the pattern seen in Fig. 35. Reactions of isolated paramecia corticeswith anti-Tn-T gives rise to a well-ordered pattern of fluorescent specks of uniformdistribution. These are believed to be due to crossreaction with basal bodies (Fig. 36).Fig. 35, bar, \1 (im; Figs 36-40, bar, 7,um.

Anti-troponin T in non-muscle cells 17

Figs 41-43.Indirect immunofluorescence of sea-urchin eggs (S. purpuratus). Anti-Tn-Tshowed intense regions of fluorescence at pericentrosomal regions of the mitotic appar-atus. A clear region (non-reactive) is seen at the centrosome (Figs 41, 42). Samples ofeggs from the same batch were also processed with anti-tubulin; the characteristicmicrotubule arrays were observed (Fig. 43). Fig. 41, bar, 19 jUm; Figs 42-43, bar, 28 jxm.

18 S.-S. Lint, G. E. Hering and G. G. Borisy

function cannot be adequately resolved. However, the extreme evolutionary persist-ence of the microtubule-associated epitope suggests conservation of a particular sitedue to some fundamental requirement in cellular function. The localization of othercrossreactive components to regions of Caz+-related activity further suggests somecommon function between microtubule-associated and non-microtubule-associatedepitopes, possibly relating to Ca2+ regulation. Further progress in this field willrequire isolation of the crossreactive species and its molecular analysis.

We are grateful to the following for gifts of cells: Drs Philippa Claude, Randall Dimond, RyokoKuriyama, Andrew Levin, Eileen Paul and Dale Vandre. We thank Dr John Fallon and MsRebecca Fuldner for use of equipment and their expertise in paraffin-section immunocyto-chemistry. 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) toS.S.L. and a National Institutes of Health grant (GM 30385) to G.G.B.

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{Received 7February 1986-Accepted 17April 1986)