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Uptake of fetal proteins by Trypanosoma cruzi immunofluorescence and ultrastructural studies
iniernational Journal for l’arasi%hgy. 1976. Vol. 6. pp. 319-386. Pergmon Press. Prinred
in
Grea!
Britain.
UPTAKE OF FETAL PROTEINS BY TRYPANOSOMA CRUZI IMMUNOFLUORESCENCE AND ULTRASTRUCTURAL STUDIES A. BRETA~A
and J. A. O’DALY
Instituto Venezolano de Investigaciones Cientifieas, Center of Microbioiogy Apartado 1827, Caracas, Venezuela (Received 26 hoverer
and Cell Biology,
1975)
Abstract-BRETAfiA
A. and O’DALY J. A. 1976. Uptake of fetal proteins by Trypanosoma cruzi. International Journalfor Parasitology 6: 379-386. Isolated proteins from fetal calf serum needed for trypanosomal growth were labelled with Pas, colloidal gold and fluorochrome tagged specific antibodies. The proteins were localized in the membranes and cytoplasm of parasites cutured in vitro in a defined liquid medium.
INDEX KEY WORDS: Trvpanosoma cruzi; uptake fetal proteins; gold Iabelled fetal proteins; ultrastructural localization of fetal proteins; eukaryote division process.
~TRODU~ON PREVIOUSLY (O’Daly, 197%~) it was reported that certain factors present in fetal calf serum stimulate division of Trypanosoma cruzi and promote the up-
take of SH thymidine by these parasites. Furthermore, the stimulation of ‘H thymidine uptake has been found to be associated with 5 serum proteins isolated from fetal calf serum by (NH,) $01 precipitation andionexchange chromatography (O’Daly, 197%). Methods for the 1abelIing and subsequent localization of some proteins in the parasite cytoplasm are presented in this paper. MATERIALS
AND METHODS
Parasite strains and their cultivation. Three strains: cruzi (Chaaas, 1909) were used. All these strains were cultivated a$ prehously published @‘Daly, 197%). Ten days’ old cultures were employed in all experiments. Preparation ofprotej~s for trypa~oso~a~growth. DEAE3 sup; DEAE3 sup, TEAE-2 and DEAEJ sup, TEAE-3 were prepared by (NH&S04 precipitation and ion exchange chromatography as published @‘Daly, 1975b). Rabbit IgG was isolated following methods previously reported (Goldstein, Slizys & Chase, 1961; Goldstein, Spalding & Hunt, 1962). Isolation of specific antibodies. Anti-DEAE-3 sup, anti-DEAE-3 sup, TEAE-2; anti-DEAE-3 sup, TEAE-3 and anti-rabbit IgG were prepared by immunizing goats with the purified proteins according to published schedules @‘Daly, 1975b). Goat specific IgG was subsequently isolated by 37% (v/v) (NHd)tSOd precipitation --
FL, Ma and Y of Trypanosoma (Schizotrypanum)
*Graduate student in the Center of Mi~obiolo~ Cell Biology.
and 379
and DEAE-cellulose chromatography (Goldstein ei aZ., 1961; Goidstein er al., 1962). Preparation of fluorescent conjugates. Specific antiDEAE-3 sup, anti-DEAE-3 sup, TEAE-2 and antirabbit IgG were conjugated with fluorescein and/or rhodamine isothiocyanate following the method of Cebra & Goldstein (1965). Radioiodination of fetal sermn proteins. Fifty-five milligrams of DEAE3 sup TEAE3 were labelled with 1 mCi of Pas (New England Nuclear, NEN, Carrier-free Iodine) followina the method of Marchalonis 09691. After ieparation-of the labelled protein with a‘ G-ii Sephadex column (Pharmacia) the exclusion pool was lyophilized, showing 6800 CPM&g of specific activity. Label&g of DEAE-3 sup with colIoidai gold. The colloidal gold was prepared by the method of Faulk & Taylor (1971), modified by Romano, Stolinski & HughesJones (1974); 250 mg of DEAE3, sup were dissolved in 20 ml of distilled Hz0 and mixed with 60 ml of colloidal gold as published (Roman0 et al., 1974). To ensure that all gold particles were adsorbed on to protein, 8 ml of NaCl 100 mg/ml (w/v) solution pH 7.0 were added. The mixture was centrifuged to 1700 g for 20 min and oc~sionally a small precipitate was found. The goldlabelled DEAE3, sup was subsequently concentrated by vacuum dialysis up to 50 mg/ml (w/v), dialyzed 24 h at 4°C against synthetic medium @‘Daly, 197.5~) and sterilized through 0.22 nm Millipore filters. Fluorescent localization of proteins. Before being assayed for its ability to enter trypanosomes, DEAE-3 sup, TEAE-2 was dialyzed against distilled Hz0 for 48 h at 4°C. The protein content of the fraction was determined by the Folin phenol method (Lowry, Rosebrounh. Farr & Randall, 1951). Subsequently each’ fraction was. diluted with the culture medium to adjust its protein content to 1 mgjml. D&ted fractions were dialyzed against the culture medium for 24 h at 4°C. After dialysis the fractions were sterilized by filtration through O-22 nm Millipore filters.
380
A. BRETAAAand J. A. O’DALY
Six million organisms from IO-day-old cultures (in exponential growth phase) were suspended in 30 ml of medium containing the protein fraction and incubated at 26°C. To determine the entrance of protein in T. cruzi cytoplasm, 0.2 ml aliquots were withdrawn from the falcon flasks (Falcon Plastics, Oxnard, U.S.A.) at 24 h intervals. The samples were spun in a cytocentrifuge (Shandon) at 900 g for 15 min. The slides were then processed for immunofluorescent staining following the method of Cebra & Goldstein (1965). Autoradiogrdphy. The radio-labelled protein DEAE-3 sup, TEAE-3 was processed as the protein used for
immunofluorescent studies. Cultures were set up in the same conditions and at day 8-10 (exponential growth phase) an equal volume of 5% v/v glutaraldehyde in synthetic medium pH 7.5 was added, incubated for 2 h at 4°C and subsequently processed for autoradiography at fine structural level as previously published (O’Daly, 1975a). Electron-microscopy
of gold-labelled trypanosomes.
@6
ml of DEAE-3 sup, TEAE-2 containing 50 mg/ml of labelled protein were diluted with 30 ml of synthetic medium (O’Daly, 1975a) to adjust its protein content to 1 mg/ml concentration. Six million organisms from IO-day-old cultures were suspended in 10 falcon flasks each containing 30 ml of culture medium and incubated at 26°C. To determine the entrance of protein in T. cruzi cytoplasm, flasks were fixed with an
equal volume of 5% v/v glutaraldehyde at 0, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 2 days and 4 days of growth respectively. 1 ml aliquot was taken for light microscopy studies, the rest being embedded in Maraglas for ultrastructural studies as explained previously (O’Daly & Bretafia, 1976). RESULTS Localization
of fetal proteins
Figures 1 and 2 show the granular fluorescence in trypanosomal cytoplasm when stained with fluorescein (Fig. 1) or rhodamine (Fig. 2) labelled specific anti-DEAE-3 sup, TEAE-2. As controls, fluorescein or rhodamine labelled 1gG were used, showing no cytoplasmic staining in any case. Furthermore, a previous adsorption of the fluorochrome labelled anti-DEAE-3 sup TEAE-2, with the specific antigen gave a total blocking of fluorescence in the parasite cytoplasm. Figures 3 and 4 correspond to the autoradiography of the 11z5labelled protein at the fine structural level. The radio-labelled protein was localized in the parasite’s cytoplasm (Fig. 3) and inside membrane bound dense granules (Fig. 4). However, the gold tagged protein gave us a better localization at ultrastructural level. In Fig. 5 after 10 min of culture electron-dense dots, corresponding to the gold containing protein can be observed at the parasite’s membrane. The same dots can be seen also inside the cytostome (Fig. 6) and in granules around the cytostome periphery (Fig. 7). After 40 min of growth, the protein is localized nonmembrane bound, in the parasite’s cytoplasm (Fig. 8) and subsequently inside granules membranebound (Fig. 9). Some granules have electron-trans-
I.J.P. VOL.6. 1976
parent matrix (Figs. 10 & ll), however, as the culture approaches the exponential phase, all the granules in the cytosplasm show a dense matrix with abundant protein inside them (Figs. 12 & 13). At nuclear division stages, some granules are seen just beside the nuclear membrane envelope (Fig. 14). Occasionally a clear communication can be observed between the granules and the perinuclear smooth endoplasmic reticulum (Fig. 16). It should be noted that the protein containing granules are still present in the cytoplasm after nuclear division (Fig. 15). After 5 min of growth, the parasite membranes, free in the medium, are covered with the goldtagged proteins (Figs. I7 & 18). Light microscopy
studies of gold labelledprotein
A better approach to discern the protein distribution in the parasite division process is the observation of the gold labelled parasites after glutaraldehyde fixation under the phase-contrast microscope. In Fig. 19 non-labelled episastigotes and trypomastigotes are presented in phase-contrast microscopy. No colour can be seen in their cytoplasms. After growing the parasites 2 days in the presence of gold tagged DEAE-3 sup protein, virtually all epimastigotes showed yellow granules inside their cytoplasm (Fig. 20). Some epimastigotes, in process of division showed an equal distribution of gold between the two daughter parasites at the beginning of division (Fig. 21, arrow) and when division was almost completed (Fig. 22, arrow). At the stationary phase of growth, trypomastigotes showed gold labelled granules at their posterior ends (Figs. 23 & 24). DISCUSSION By using 3 different methodologies, i.e. immunofluorescence, autoradiography and gold-labelled proteins we have followed through the parasite organelles the localization of a protein needed for trypanosome division and growth. The method of choice was the protein labelling with colloidal gold, which permitted a clear definition of the protein pathway through membranes and cytoplasm at the fine structural level. At early time intervals the protein was adsorbed on to the parasite membranes and was also found inside the cytostome. At later time intervals the protein could be observed in the cytoplasmic matrix. After 40 min all parasites had abundant granules containing protein in their cytoplasms. These granules might well be phagolysozomes where the protein is degraded, which suggests many questions on the mechanism of action of the protein. The binding of the protein to the membrane surface also suggests that a signal at this level can start trypanosome division, as is the case with antigens and lectins on lymphocytes (Greanes & Janossy, 1972).
I.J:P. VOL.6. 1976
Uptake of fetal proteins by Trypanosoma cruzi
On the other hand, the presence of granules containing protein at the nuclear periphery might indicate that protein products could be transported to the nucleus to start parasite division. The presence of trypanosomal membranes, free in the medium @‘Daly & Bretana, 1976) covered by gold-tagged protein molecules emphasizes the possibility that protein of host origin ,covers specific determinants on the trypanosomal surface enabling them to avoid the host immunological response. The protein as seen with the phas~contrast microscope is apparently divided equally among the daughter cells after parasite division. Once differentiation to trypomastigotes is completed, a protein containing granule can be observed at the parasite posterior end. As far as we know this is the first account of localization of a protein at fine structural level involved in the division process of eukaryotic
organisms.
~~k~l~wledgements-be thank Mrs. DelValle Perales for helping with the manuscript.
Russo
REFERENCES CEBRA, J. J. & GOLDSTEING. 1965. Chromato~aphic purification of tetramethyl rhodamine-immunoglobulin conjugates and their use in the cellular localization of rabbit ~-g~obuljn polypeptide chains. ~~~r~aZ of immunology 95: 230-345. FAULK W. P. & TAYLORG. M, 1971. An immunocolloid method for the electron microscope. immunochemistry 8: 1081-1084.
381
GOLDSTEING., SLIZYSI. S. & CHASEM. W. 1961. Studies on fluorescent antibody staining. Journal of Experimental Medicine 114: 89-109. GOLDSTEING., SPALDINGB. H, & RUNT W. B. 1962. Studies on fluorescent antibody staining. II. Inhibition by sub-optimally conjugated antibody globulins. Proceedings of the Society for ~xperimentai Biology and Medicine 1I1 : 416-42 1. GREANESM. F. & JANOSSYG. 1972. Elicitation of selective T and B lymphocyte response by cell surface binding ligands. Transpl~fatian Review 11: 87-130. LOWRY O.,R~SEBROUGHN. J., FARR A. L. & RANDALL R. 1951. Protein measurement with folin nbenoi reagents. JaurnaI of 3~ologi~a~ Chemistry 193: 26.5276. MARCHALONZS J. J. 1969. An enzymic method for the iodination of immunoglobulins and other proteins. 3io~hem~cal Journal 113: 299-305. O’DALY J. A. 1975a. A new liquid medium for Trypanosoma (Schizotrypanum) cruzi. Journal of Protozoology 22: 265-270. O’DALY J. A. 197%. Serum proteins promoting %I thymidine uptake by Trypanosoma (Schizotrypanum) cruzi (Chagas) in vitro. Journal of Protozoology 22: 550-55s. O’DALY J. A. & BRIXTA~AA. 1976. Ultr~tructu~l observations of Trypanosoma crazi in a liquid medium. The kinetoplast-mitochondrion in division forms. Internationai Journal of Parasitology. In press. ROMANO E. L., STOLINSKIC. & HUGHES-JONESN. C. 1974.An antigIobuiin reagent iabelled with colloidal gold for use in electron microscopy. Immunochemistry
11: 521422.
FIG. I. Tmmunofluorescein FIG. 2. Immunorhodamine FIG. 3. Ei~tronmicro~ph FIG. 4. Electronmicrograph
stained epimastigotes at early exponential growth phase. Numerous granules can be seen inside the parasite cytoplasm. stained epimastigotes in advanced exponential growth phase. Virtually all parasites show numerous granules inside the cytoplasm. of sections through 1x26 protein labelled parasites. The labelfed protein can be observed in the parasite cytoplasm. m: mitochondria. of sections through I 125protein labelled epimastigotes. The labelled protein is seen inside a membrane-bound granule (g). m: mitochondria.
FIG. 5. Cross-section
of two epimastigotes with numerous electron-dense dots (arrow) belonging to the gold-tagged protein on the membrane surface after 5 min of culture. mb: membranes. FIG. 6. Section showing epimastigote with abundant electron-dense dots inside the cytostome (Cy) after 5 min of culture. Fro. 7. Cross-section showing epimastigote with dots inside the cytostome (Cy) and in granules (g) at the cystostome periphery. Fro. 8. Cross-section of a parasite at 20 min after culture. Abundant el~tron~dense dots, belonging to the goId-tagged protein can be observed free in the cytoplasm (arrows). k: kinetoplast.
FIG. 9. Epimastigote in advanced exponential growth phase (4 days) cultured in the presence of the gold-tagged protein. Numerous electron dense granules (g) with gold-labelled proteins within them can be observed. k: kinetoplast. FIG. 10. Cross-section of epimastigote 40 min after culture with the gold-tagged protein inside a membrane-bound granule which contains a clear matrix. FIG. 11. High-power view of granules (g) containing gold-labellcd protein at early exponential growth phase. A clear matrix, predominates in most parasite granules. N: nucleus. FIG. 12. High-power view of granules (g) containing gold-labelled protein at late time intervals (4 days) of culture. A dense matrix predominates in most parasite granules.
FIG. 13. Low-power view of parasites with gold-labeiled proteins after 4 days of culture. All parasites show granules (g)
with a dense matrix. N: nucleus; n: nucleolus; k: kinetoplast. Fm. 14. Cross-section of epimastigote in nuclear division with abundant tubules (t) within the nuclear matrix. A granule (g) containing gold-tagged protein can be observed adjacent to the nuclear membrane. N: nucleus. FIG. 15. Cross-section of epimastigote after nuclear division with abundant granules (g) containing protein in the cytoplasm. k: kinetoplast; N: nucleus; n: nucteolus.
I^ ._ .
___--. _--^
_.,-
. ..-
.-
._.
,”
._
___._~~
__-.__ _ _
_
I
FIG. 16. High magnification of epimastigote in nuclear division with tubules (t) within the nuclear matrix. A granule (g) with gold-tagged protein can be observed adjacent to the perinuclear space (double arrow). N: nucleus. FIG. 17. High magnification of membranes (mb) free in the culture medium. Numerous electron-dense dots corresponding to gold-tagged proteins (arrows) cover the membrane surface. FIG. 18. Cross-section of a parasite with adjacent membranes (mb) containing gold-tagged protein molecules. The electron-dense dots can be observed in the main membrane axis and in branches (arrows) perpendicularly directed towards the membrane axis.
FIGS. 19-24. Trypanosomacmzi growing in synthetic medium containing I)EAE-3 sup gold-lab&led protein. In Fig. 19
epimastigot~
(Ep) as seen under the phase contrast microscope cultured with the non-la~lled protein. No colour can be observed in their cytoplasms. In Figs. 20-22 all parasites at exponential growth phase show yellow granules inside the cytoplasm. Epimastigotes (Ep) in process of division can be seen, with equally distributed protein granules among daughter cells (arrows). In Figs. 23 & 24 trypomastigotes are presented as the culture enters the stationary phase of growth. At their posterior ends a granule, corresponding to gold-labelled protein can be discerned (arrow). f.p. 386
in
Grea!
Britain.
UPTAKE OF FETAL PROTEINS BY TRYPANOSOMA CRUZI IMMUNOFLUORESCENCE AND ULTRASTRUCTURAL STUDIES A. BRETA~A
and J. A. O’DALY
Instituto Venezolano de Investigaciones Cientifieas, Center of Microbioiogy Apartado 1827, Caracas, Venezuela (Received 26 hoverer
and Cell Biology,
1975)
Abstract-BRETAfiA
A. and O’DALY J. A. 1976. Uptake of fetal proteins by Trypanosoma cruzi. International Journalfor Parasitology 6: 379-386. Isolated proteins from fetal calf serum needed for trypanosomal growth were labelled with Pas, colloidal gold and fluorochrome tagged specific antibodies. The proteins were localized in the membranes and cytoplasm of parasites cutured in vitro in a defined liquid medium.
INDEX KEY WORDS: Trvpanosoma cruzi; uptake fetal proteins; gold Iabelled fetal proteins; ultrastructural localization of fetal proteins; eukaryote division process.
~TRODU~ON PREVIOUSLY (O’Daly, 197%~) it was reported that certain factors present in fetal calf serum stimulate division of Trypanosoma cruzi and promote the up-
take of SH thymidine by these parasites. Furthermore, the stimulation of ‘H thymidine uptake has been found to be associated with 5 serum proteins isolated from fetal calf serum by (NH,) $01 precipitation andionexchange chromatography (O’Daly, 197%). Methods for the 1abelIing and subsequent localization of some proteins in the parasite cytoplasm are presented in this paper. MATERIALS
AND METHODS
Parasite strains and their cultivation. Three strains: cruzi (Chaaas, 1909) were used. All these strains were cultivated a$ prehously published @‘Daly, 197%). Ten days’ old cultures were employed in all experiments. Preparation ofprotej~s for trypa~oso~a~growth. DEAE3 sup; DEAE3 sup, TEAE-2 and DEAEJ sup, TEAE-3 were prepared by (NH&S04 precipitation and ion exchange chromatography as published @‘Daly, 1975b). Rabbit IgG was isolated following methods previously reported (Goldstein, Slizys & Chase, 1961; Goldstein, Spalding & Hunt, 1962). Isolation of specific antibodies. Anti-DEAE-3 sup, anti-DEAE-3 sup, TEAE-2; anti-DEAE-3 sup, TEAE-3 and anti-rabbit IgG were prepared by immunizing goats with the purified proteins according to published schedules @‘Daly, 1975b). Goat specific IgG was subsequently isolated by 37% (v/v) (NHd)tSOd precipitation --
FL, Ma and Y of Trypanosoma (Schizotrypanum)
*Graduate student in the Center of Mi~obiolo~ Cell Biology.
and 379
and DEAE-cellulose chromatography (Goldstein ei aZ., 1961; Goidstein er al., 1962). Preparation of fluorescent conjugates. Specific antiDEAE-3 sup, anti-DEAE-3 sup, TEAE-2 and antirabbit IgG were conjugated with fluorescein and/or rhodamine isothiocyanate following the method of Cebra & Goldstein (1965). Radioiodination of fetal sermn proteins. Fifty-five milligrams of DEAE3 sup TEAE3 were labelled with 1 mCi of Pas (New England Nuclear, NEN, Carrier-free Iodine) followina the method of Marchalonis 09691. After ieparation-of the labelled protein with a‘ G-ii Sephadex column (Pharmacia) the exclusion pool was lyophilized, showing 6800 CPM&g of specific activity. Label&g of DEAE-3 sup with colIoidai gold. The colloidal gold was prepared by the method of Faulk & Taylor (1971), modified by Romano, Stolinski & HughesJones (1974); 250 mg of DEAE3, sup were dissolved in 20 ml of distilled Hz0 and mixed with 60 ml of colloidal gold as published (Roman0 et al., 1974). To ensure that all gold particles were adsorbed on to protein, 8 ml of NaCl 100 mg/ml (w/v) solution pH 7.0 were added. The mixture was centrifuged to 1700 g for 20 min and oc~sionally a small precipitate was found. The goldlabelled DEAE3, sup was subsequently concentrated by vacuum dialysis up to 50 mg/ml (w/v), dialyzed 24 h at 4°C against synthetic medium @‘Daly, 197.5~) and sterilized through 0.22 nm Millipore filters. Fluorescent localization of proteins. Before being assayed for its ability to enter trypanosomes, DEAE-3 sup, TEAE-2 was dialyzed against distilled Hz0 for 48 h at 4°C. The protein content of the fraction was determined by the Folin phenol method (Lowry, Rosebrounh. Farr & Randall, 1951). Subsequently each’ fraction was. diluted with the culture medium to adjust its protein content to 1 mgjml. D&ted fractions were dialyzed against the culture medium for 24 h at 4°C. After dialysis the fractions were sterilized by filtration through O-22 nm Millipore filters.
380
A. BRETAAAand J. A. O’DALY
Six million organisms from IO-day-old cultures (in exponential growth phase) were suspended in 30 ml of medium containing the protein fraction and incubated at 26°C. To determine the entrance of protein in T. cruzi cytoplasm, 0.2 ml aliquots were withdrawn from the falcon flasks (Falcon Plastics, Oxnard, U.S.A.) at 24 h intervals. The samples were spun in a cytocentrifuge (Shandon) at 900 g for 15 min. The slides were then processed for immunofluorescent staining following the method of Cebra & Goldstein (1965). Autoradiogrdphy. The radio-labelled protein DEAE-3 sup, TEAE-3 was processed as the protein used for
immunofluorescent studies. Cultures were set up in the same conditions and at day 8-10 (exponential growth phase) an equal volume of 5% v/v glutaraldehyde in synthetic medium pH 7.5 was added, incubated for 2 h at 4°C and subsequently processed for autoradiography at fine structural level as previously published (O’Daly, 1975a). Electron-microscopy
of gold-labelled trypanosomes.
@6
ml of DEAE-3 sup, TEAE-2 containing 50 mg/ml of labelled protein were diluted with 30 ml of synthetic medium (O’Daly, 1975a) to adjust its protein content to 1 mg/ml concentration. Six million organisms from IO-day-old cultures were suspended in 10 falcon flasks each containing 30 ml of culture medium and incubated at 26°C. To determine the entrance of protein in T. cruzi cytoplasm, flasks were fixed with an
equal volume of 5% v/v glutaraldehyde at 0, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 2 days and 4 days of growth respectively. 1 ml aliquot was taken for light microscopy studies, the rest being embedded in Maraglas for ultrastructural studies as explained previously (O’Daly & Bretafia, 1976). RESULTS Localization
of fetal proteins
Figures 1 and 2 show the granular fluorescence in trypanosomal cytoplasm when stained with fluorescein (Fig. 1) or rhodamine (Fig. 2) labelled specific anti-DEAE-3 sup, TEAE-2. As controls, fluorescein or rhodamine labelled 1gG were used, showing no cytoplasmic staining in any case. Furthermore, a previous adsorption of the fluorochrome labelled anti-DEAE-3 sup TEAE-2, with the specific antigen gave a total blocking of fluorescence in the parasite cytoplasm. Figures 3 and 4 correspond to the autoradiography of the 11z5labelled protein at the fine structural level. The radio-labelled protein was localized in the parasite’s cytoplasm (Fig. 3) and inside membrane bound dense granules (Fig. 4). However, the gold tagged protein gave us a better localization at ultrastructural level. In Fig. 5 after 10 min of culture electron-dense dots, corresponding to the gold containing protein can be observed at the parasite’s membrane. The same dots can be seen also inside the cytostome (Fig. 6) and in granules around the cytostome periphery (Fig. 7). After 40 min of growth, the protein is localized nonmembrane bound, in the parasite’s cytoplasm (Fig. 8) and subsequently inside granules membranebound (Fig. 9). Some granules have electron-trans-
I.J.P. VOL.6. 1976
parent matrix (Figs. 10 & ll), however, as the culture approaches the exponential phase, all the granules in the cytosplasm show a dense matrix with abundant protein inside them (Figs. 12 & 13). At nuclear division stages, some granules are seen just beside the nuclear membrane envelope (Fig. 14). Occasionally a clear communication can be observed between the granules and the perinuclear smooth endoplasmic reticulum (Fig. 16). It should be noted that the protein containing granules are still present in the cytoplasm after nuclear division (Fig. 15). After 5 min of growth, the parasite membranes, free in the medium, are covered with the goldtagged proteins (Figs. I7 & 18). Light microscopy
studies of gold labelledprotein
A better approach to discern the protein distribution in the parasite division process is the observation of the gold labelled parasites after glutaraldehyde fixation under the phase-contrast microscope. In Fig. 19 non-labelled episastigotes and trypomastigotes are presented in phase-contrast microscopy. No colour can be seen in their cytoplasms. After growing the parasites 2 days in the presence of gold tagged DEAE-3 sup protein, virtually all epimastigotes showed yellow granules inside their cytoplasm (Fig. 20). Some epimastigotes, in process of division showed an equal distribution of gold between the two daughter parasites at the beginning of division (Fig. 21, arrow) and when division was almost completed (Fig. 22, arrow). At the stationary phase of growth, trypomastigotes showed gold labelled granules at their posterior ends (Figs. 23 & 24). DISCUSSION By using 3 different methodologies, i.e. immunofluorescence, autoradiography and gold-labelled proteins we have followed through the parasite organelles the localization of a protein needed for trypanosome division and growth. The method of choice was the protein labelling with colloidal gold, which permitted a clear definition of the protein pathway through membranes and cytoplasm at the fine structural level. At early time intervals the protein was adsorbed on to the parasite membranes and was also found inside the cytostome. At later time intervals the protein could be observed in the cytoplasmic matrix. After 40 min all parasites had abundant granules containing protein in their cytoplasms. These granules might well be phagolysozomes where the protein is degraded, which suggests many questions on the mechanism of action of the protein. The binding of the protein to the membrane surface also suggests that a signal at this level can start trypanosome division, as is the case with antigens and lectins on lymphocytes (Greanes & Janossy, 1972).
I.J:P. VOL.6. 1976
Uptake of fetal proteins by Trypanosoma cruzi
On the other hand, the presence of granules containing protein at the nuclear periphery might indicate that protein products could be transported to the nucleus to start parasite division. The presence of trypanosomal membranes, free in the medium @‘Daly & Bretana, 1976) covered by gold-tagged protein molecules emphasizes the possibility that protein of host origin ,covers specific determinants on the trypanosomal surface enabling them to avoid the host immunological response. The protein as seen with the phas~contrast microscope is apparently divided equally among the daughter cells after parasite division. Once differentiation to trypomastigotes is completed, a protein containing granule can be observed at the parasite posterior end. As far as we know this is the first account of localization of a protein at fine structural level involved in the division process of eukaryotic
organisms.
~~k~l~wledgements-be thank Mrs. DelValle Perales for helping with the manuscript.
Russo
REFERENCES CEBRA, J. J. & GOLDSTEING. 1965. Chromato~aphic purification of tetramethyl rhodamine-immunoglobulin conjugates and their use in the cellular localization of rabbit ~-g~obuljn polypeptide chains. ~~~r~aZ of immunology 95: 230-345. FAULK W. P. & TAYLORG. M, 1971. An immunocolloid method for the electron microscope. immunochemistry 8: 1081-1084.
381
GOLDSTEING., SLIZYSI. S. & CHASEM. W. 1961. Studies on fluorescent antibody staining. Journal of Experimental Medicine 114: 89-109. GOLDSTEING., SPALDINGB. H, & RUNT W. B. 1962. Studies on fluorescent antibody staining. II. Inhibition by sub-optimally conjugated antibody globulins. Proceedings of the Society for ~xperimentai Biology and Medicine 1I1 : 416-42 1. GREANESM. F. & JANOSSYG. 1972. Elicitation of selective T and B lymphocyte response by cell surface binding ligands. Transpl~fatian Review 11: 87-130. LOWRY O.,R~SEBROUGHN. J., FARR A. L. & RANDALL R. 1951. Protein measurement with folin nbenoi reagents. JaurnaI of 3~ologi~a~ Chemistry 193: 26.5276. MARCHALONZS J. J. 1969. An enzymic method for the iodination of immunoglobulins and other proteins. 3io~hem~cal Journal 113: 299-305. O’DALY J. A. 1975a. A new liquid medium for Trypanosoma (Schizotrypanum) cruzi. Journal of Protozoology 22: 265-270. O’DALY J. A. 197%. Serum proteins promoting %I thymidine uptake by Trypanosoma (Schizotrypanum) cruzi (Chagas) in vitro. Journal of Protozoology 22: 550-55s. O’DALY J. A. & BRIXTA~AA. 1976. Ultr~tructu~l observations of Trypanosoma crazi in a liquid medium. The kinetoplast-mitochondrion in division forms. Internationai Journal of Parasitology. In press. ROMANO E. L., STOLINSKIC. & HUGHES-JONESN. C. 1974.An antigIobuiin reagent iabelled with colloidal gold for use in electron microscopy. Immunochemistry
11: 521422.
FIG. I. Tmmunofluorescein FIG. 2. Immunorhodamine FIG. 3. Ei~tronmicro~ph FIG. 4. Electronmicrograph
stained epimastigotes at early exponential growth phase. Numerous granules can be seen inside the parasite cytoplasm. stained epimastigotes in advanced exponential growth phase. Virtually all parasites show numerous granules inside the cytoplasm. of sections through 1x26 protein labelled parasites. The labelfed protein can be observed in the parasite cytoplasm. m: mitochondria. of sections through I 125protein labelled epimastigotes. The labelled protein is seen inside a membrane-bound granule (g). m: mitochondria.
FIG. 5. Cross-section
of two epimastigotes with numerous electron-dense dots (arrow) belonging to the gold-tagged protein on the membrane surface after 5 min of culture. mb: membranes. FIG. 6. Section showing epimastigote with abundant electron-dense dots inside the cytostome (Cy) after 5 min of culture. Fro. 7. Cross-section showing epimastigote with dots inside the cytostome (Cy) and in granules (g) at the cystostome periphery. Fro. 8. Cross-section of a parasite at 20 min after culture. Abundant el~tron~dense dots, belonging to the goId-tagged protein can be observed free in the cytoplasm (arrows). k: kinetoplast.
FIG. 9. Epimastigote in advanced exponential growth phase (4 days) cultured in the presence of the gold-tagged protein. Numerous electron dense granules (g) with gold-labelled proteins within them can be observed. k: kinetoplast. FIG. 10. Cross-section of epimastigote 40 min after culture with the gold-tagged protein inside a membrane-bound granule which contains a clear matrix. FIG. 11. High-power view of granules (g) containing gold-labellcd protein at early exponential growth phase. A clear matrix, predominates in most parasite granules. N: nucleus. FIG. 12. High-power view of granules (g) containing gold-labelled protein at late time intervals (4 days) of culture. A dense matrix predominates in most parasite granules.
FIG. 13. Low-power view of parasites with gold-labeiled proteins after 4 days of culture. All parasites show granules (g)
with a dense matrix. N: nucleus; n: nucleolus; k: kinetoplast. Fm. 14. Cross-section of epimastigote in nuclear division with abundant tubules (t) within the nuclear matrix. A granule (g) containing gold-tagged protein can be observed adjacent to the nuclear membrane. N: nucleus. FIG. 15. Cross-section of epimastigote after nuclear division with abundant granules (g) containing protein in the cytoplasm. k: kinetoplast; N: nucleus; n: nucteolus.
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FIG. 16. High magnification of epimastigote in nuclear division with tubules (t) within the nuclear matrix. A granule (g) with gold-tagged protein can be observed adjacent to the perinuclear space (double arrow). N: nucleus. FIG. 17. High magnification of membranes (mb) free in the culture medium. Numerous electron-dense dots corresponding to gold-tagged proteins (arrows) cover the membrane surface. FIG. 18. Cross-section of a parasite with adjacent membranes (mb) containing gold-tagged protein molecules. The electron-dense dots can be observed in the main membrane axis and in branches (arrows) perpendicularly directed towards the membrane axis.
FIGS. 19-24. Trypanosomacmzi growing in synthetic medium containing I)EAE-3 sup gold-lab&led protein. In Fig. 19
epimastigot~
(Ep) as seen under the phase contrast microscope cultured with the non-la~lled protein. No colour can be observed in their cytoplasms. In Figs. 20-22 all parasites at exponential growth phase show yellow granules inside the cytoplasm. Epimastigotes (Ep) in process of division can be seen, with equally distributed protein granules among daughter cells (arrows). In Figs. 23 & 24 trypomastigotes are presented as the culture enters the stationary phase of growth. At their posterior ends a granule, corresponding to gold-labelled protein can be discerned (arrow). f.p. 386