0022-1554/78/2605-0349/$02.00/O THE

JOURNAL

OF

HISTOCHEMISTRY

© 1978 by The

Copyright

AND

Histochemical

Vol. 26, No. 5, pp. 349-358, 1978 Printed in U.S.A.

CYTOCHEMISTRY

Society,

Inc.

ULTRASTRUCTURAL

LOCALIZATION

OF

TRYPANOSOMA THAIS Instituto

de Biofisica,

SOUTO-PADRON

Universidade

AND

Federal

do

Rio

for publication

August

WANDERLEY

de Janeiro,

de Janeiro,

Received

BASIC

PROTEINS

IN

CRUZF

2O,’XX,

DE

Cidade

SOUZA

Universit#{225}ria,

Il/ia

do

Fund#{227}o, Rio

Brasil

2, 1977, and in revised

form

December

13, 1977 (MS

77-181)

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (EPTA) techniques were applied in epimastigote and trypomastigote forms of the pathogenic protozoa Trypanosoma cruzi to detect basic proteins at the ultrastructural level. With both techniques, reaction was observed in the nucleus and in some cytoplasmic vacuoles. In the kinetoplast of epimastigotes, reaction was observed only at its periphery. In trypomastigotes, however, an intense reaction was observed in the spherical kinetoplast. With the ethanolic phosphotungstic acid technique, reaction was also observed in ribosomes and at the peripheral doublet microtubules of the flagellum. The filaments which form the paraflagellar structure did not react. Cytochemical

tron

methods

microscopy

have

used

proteins (mainly histones). (20) described a postformalin (AS) plied

staining method by some authors

tungstic

acid

solutions

(PTA)

for

acids special

which (16, has

has 19,

been

substances

rich 25).

These

basic

basic

published

ical

proteins

are

tural

and

bases

tigote

reg-

agent host

on cytochemin protozoa

are

and

identified

of

of natural cells. In

results

obtained

by

family which include diseases such as Chagas and Both have a high prevalence and Africa, respectively. This

methods structural

group since

is also have

Microorganism: (liver infusion-tryptose) ing from 3 to 8 days

about 20% this structure We

thought,

est

to

teins

of the (for

cellular a review

therefore,

investigate

in trypanosomatids,

the

that

a condensation of in its unique mitocruzi, for example, DNA see

localization

by using

as

is localized in Simpson (27)).

it would

be of

to locate level.

the

source

forms.

of

of trypomastigotes.

gote

and

using

from

pro-

‘This work has been supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnol#{243}gico (CNPq), Conselho de Ensino para Graduados da U.F.R.J. and Financiadora de Estudos e Projetos (F!NEP-FNDCT-314/CT).

amas-

to penetrate we describe and

the

at

PTA

the

ultra-

METHODS

and

The forms

was

separate

a modification

as described

column

by Goldberg

microscopy

8-day

cultures

percentage

To

DEAE-cellulose (17)

AS

proteins

AND

examination.

Electron

the

T. cruzi was cultivated is LIT medium (7) for periods varyat 27#{176}C. We used 3-day cultures

trypomastigotes,

ployed

cytochemical

the

basic

trypomastigote

Godfrey’s

epimas-

Only

forms are able to divide. form plays a fundamental of T. cruzi since it is the

of epimastigotes

that

croscopic

of inter-

basic

as amastigote,

MATERIALS

of biological importance a special structure, the

kinetoplast, which represents DNA in a definite position chondrion. In Trypanosoma

a process epimastigotes

infection and is able the present report

the Trypanosomatidae agents of important sleeping sickness. in Latin America of protozoa its members

it undergoes when

trypomastigote

tigote and epimastigote The trypomastigote role in the life cycle

of

involved

differentiation

of basic

or are

the agent of Chagas of the family Try-

transform into trypomastigotes. T. cruzi presents during its life cycle in the vertebrate host various developmental stages which on struc-

amino-

studies

proteins

panosomatidae, since cellular differentiation

been apPhosphoproteins

in

We chose T. cruzi, as a representative

methods. disease,

in alcoholic

basic

in the control of cellular ulation of gene function. Few papers have been localization

then 20).

used

of

(5, 6, 13, 24, interest since

elecbasic

MacRae and Meetz ammoniacal-silver

localization

proteinaceous

with to localize

associated

been

and

as

of epimasti-

estimated

by

mi-

epimastigotes of Lanham method

was

and em-

et al. (12).

cytochemistry:

The

1500 g for 10 mm at 4#{176} C and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 1 hr at room temperature. After fixation they were washed in buffer, post-fixed is 1% Os04 in phosphate buffer for 1 hr at room trypanosomes

were

centrifuged

349

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at

SOUTO-PADRON

350

AND

temperature, dehydrated in ethanol and embedded in Epon. Ultrathin sections were obtained with an LKB Ultratome III (LKB Instr. Co. Rockville, Md.) ultramicrotome and examined unstained or after staining with uranyl acetate or lead citrate or both, in an AEI EM6-B electron microscope. The phosphotungstic acid method (E-PTA) was employed as described by Gordon and Bensch (13). After glutaraldehyde fixation, the cells were dehydrated (without post-fixation in OsO4) in ethanol and incubated for 2 hr at room temperature in 2% PTA (Sigma Chemical Company, St. Louis, Mo.) in absolute ethanol after which time they were washed in ethanol and embedded in Epon. The ammoniacal silver (AS) method was used as described by MacRae and Meetz (20). After fixation, the cells were thoroughly washed in distilled water and incubated for 5 mis at room temperature in the AS solution. This solution was prepared just before use by gradual addition of 10% silver nitrate solution (Matheson Coleman & Bell, N.Y., N.Y.) to concentrated ammonium hydroxide until a slight turbidity appears and persists. After incubation in this solution, the cells were washed in distilled water and placed for 5 mm in a 3% formaldehyde solution, where a brown coloration appeared. After washing in distilled water they were post-fixed in 0sO, dehydrated in ethanol and embedded in Epon. RESULTS

General structure: The general structure of epimastigote and trypomastigote forms of T. cruzi from acellular cultures has been described in previous papers (9, 10). The cell is surrounded by a continuous trilaminate unit membrane. Under can

the cell be seen.

of the cell vided with ter of the in contact

membrane, The nucleus

has a typical nuclear envelope pores. A nucleolus occupies the nucleus with

the

shape surface

structural

cruzi

center of the nucleus (Fig. the nucleus has a more or Since the trypomastigotes the nucleus also has an

being in some regions close to (Fig. 1). The most striking ultra-

feature

is the

procen-

while condensed chromatin is nuclear envelope and often

extends towards the 2). In epimastigotes less spherical shape. are much elongated, elongated the cell

a row of microtubules situated in the center

of

arrangement

the

trypomastigote

of DNA

of

fibrils

T.

which

form the kinetoplast. In the epimastigotes, the kinetoplast is rod-shaped and its DNA appears filamentous and more or less tightly coiled (Fig. 2). The filaments are oriented parallel to the long axis kinetoplast ture, ments

of the cell. appears

less electron-dense, more dispersed

In the trypomastigote, as a large spherical and with (Fig. 1). When

the strucDNA filawe used 3-

DE SOUZA

day cultures, 8-day cultures ate ary

stages, cultures

only epimastigotes were found. both forms, as well as intermedi-

were observed. By passage through a DEAE-cellulose

trypomastigotes

were

isolated.

In

of stationcolumn

However,

as can

be seen in Figure 1, some cells have a kinetoplast whose structure is typical for epimastigote forms. In epimastigotes as well as in trypomastigotes,

some

plasm.

vacuoles

They

are

appear

moderately nosomatids,

dense the

either material. paraflagellar

in

or contain

As

the

in other structure

of T. cruzi method:

served in the flagellum Ammoniacal-silver cation of the had a brown

found empty

cytoa

trypais ob-

(Fig. 3). After appli-

AS reaction, the suspension to black color which could

of cells also be

seen by light microscopy, plitude contrast method

mainly associated

ski optics microscope,

(Fig. 4). In the electron appears in the form of

(1)

was used the reaction

when with

the amNomar-

electron opaque particles whose diameter measures about 56 nm. They are found either isolated or in contact with each other. In epimastigotes obtained from 3-day cultures, electron-opaque in the nucleus were observed

particles were observed (Figs. 5 and 6). A few in cytoplasmic vacuoles,

mainly

particles free in

the cytoplasm or in the kinetoplast (Figs. 5 and 6). In the last structure they were observed only at the periphery and not in the center of the mass of DNA fibrils even when the kinetoplast was in division (Fig. 6). In trypomastigotes, a large number of silver particles elongated nucleus (Fig. 7) and kinetoplast where they appear the

were seen in the in the spherical in the center of

mass

of DNA fibrils (Figs. 8 and 9). In the of the transition forms between epimastigotes and trypomastigotes, particles can also be found that are associated with the DNA kinetoplast

fibrils (Fig. 8). Phosphottmgstic

acid

ment

with

phosphotungstic

PTA) action.

gives a homogenously After application

cells obtained

ethanolic

appear

well from

observed in the the cytoplasmic some tion those

The

treat-

acid

electron-dense of this technique,

preserved.

3-day

cultures,

In

(Erethe

epimastigotes a reaction

was

nucleus (Figs. 10 and 11) and in granules. The ribosomes and

microtubules (Figs. 10-13). of the

method:

also showed a positive reacAmong the microtubules,

cytostome

(Fig.

10) and

the

eral doublets of the flagellum (Figs. 10, 13) react well. No reaction was observed

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periph12 and in the

FIG. 1. General aspect of trypanosomes isolated column. Most of the trypanosomes are trypomastigotes a kinetoplast typical for epimastigotes (K2) is also elongated being close to the cell surface. x22,500. FIG. 2. Epimastigote obtained from 3-day culture. FIG. 3. Cross section through the anterior microtubules (small arrows). The flagellum, trypomastigote body. In the upper parasite (curved arrow). x45,000.

by passage of a 10-day culture through DEAE-cellulose which have a spherical kinetoplast (Ki). A cell showing observed. The nucleus (N) of the trypomastigote form is The

kinetoplast

(K) is rod-shaped.

N, nucleus.

x 19,000.

region of a trypomastigote (T) showing the sub-pellicular showing the paraflagellar structure (PS), is attached to the a longitudinal section of a sub-pellicular microtubule is shown 351

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.1 C,

b

..

FIG.

formalin

4.

Photomicrograph ammoniacal

silver

(amplitude method. The

contrast, reaction

Nomarski optics) of epimastigotes is seen mainly in the nuclei. x600. 5) or in division (Fig. 6) subjected

FIG. 5-6. Epimastigotes in interphase (Fig. method. Granules are observed in the nucleus, in some kinetoplast (K). Figure 5, x37,500; Figure 6, x45,000.

cytoplasmic

vesicles

subjected to

(V) and

352

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the

at the

to

ammoniacal

periphery

the

postsilver

of the

BASIC

FIG. 7-9. (Fig. 7) and

Figures

Trypomastigotes in the kinetoplast

7-8, x45,000;

central

pair

in

filaments

the

the

which

kinetoplast

the cells

DNA which

mastigotes had

that

fibrils were by means

a kinetoplast

flagellum

the

paraflagellar

13). Typical trypomastireaction in all fibrils appears (Figs.

it

of the form

which body

makes

difficult isolated

as

an

electron-

14, 16).

The

reaction

the

353

CRUZI

silver method. Granules are shown in Figure 8 is from

a structure

14,

or

epimastigotes. was observed

of

electron-dense

tures,

seen in the nucleus a form in transition.

of

16). with

Some trypo-

column, typical

their

However, in contrast in epimastigotes from kinetoplasts (Fig.

appeared

with 3-day

what cul-

uniformly

15).

DISCUSSION

visualization

(Figs. together

of DEAE-cellulose with

TRYPANOSOMA

9, x60,000.

of microtubules

dense spherical is so intense

IN

subjected to the ammoniacal (Figs. 8 and 9). The kinetoplast

Figure

structure (Figs. 12 and gotes showed a uniform

PROTEINS

for

Various used which alkaline tidine,

cytochemical

to localize basic is most frequently dye lysine

methods

have

been

proteins in cells. The one applied is based on the

binding to the basic and arginine residues

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groups of hisin the poly-

354

SOUTO-PADRON

AND

DE

SOUZA

‘. ‘I

4

.*.s

.1

‘4 -‘.‘I

6 .,

Cv

;

.

a

-..

10

‘

4

1j

‘4% .6

p.

:

12

-.-

#.i,

I

-

t

.?,‘.I -I.

i

,.*.

*

‘ ._i

‘P’t,.

--..p .‘

a

,

#{149} ,

d

*

-

‘I

-.

-. ,,

,.

Is’

.

-,

:6

13

Downloaded from jhc.sagepub.com at IOWA TESTING PROGRAMS on March 16, 2015

BASIC

PROTEINS

IN

TRYPANOSOMA

355

CRUZI

.1:

‘p

f

I..

L

.l(.

.-..‘ -

acid.

Reaction

trypomastigotes. x60,000; Figure

is

observed

A kinetoplast 16, x45,000.

in

‘-“I’

rmeansofa. nucleus (N), in typical for epimastigotes

the

cytoplasmic (Fig.

15)

vesicles

(V)

also

reacts.

and

in

Figure

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the

kinetoplast 14, x 15,000;

(K)

Figure

of

15,

356

SOUTO-PADRON

peptide place,

chains. DNA has

erate

the

dude blue

fast (23).

vations

basic

Before to be

by

will

Ammoniacal

technique Black gations histones

this reaction can take removed in order to lib-

groups.

The

acid

dyes

used

in-

green, dansyl, eosin and bromphenol All these methods are used for obserlight

microscopy.

have been used to electron microscopic which

AND

basic level,

be discussed silver: was

Two

detect

techniques

proteins at the E-PTA and AS,

separately. The postformalin

introduced

in light

whereas

the

black

staining

with

and

argimne

introduced and Meetz

residues to electron (20) which

used it to localize histones in the erythropoietic cells of the chick. In that study it was shown that stem cells and early erythroblasts exhibit little reaction while small basophilic erythroblasts, polychromatophilic erythrocytes and reticulocytes exhibit an increasing amount of silver granules as maturation proceeds. The same technique was also used to localize histones in erythroid of types MacRae nuclear roid In pears

precursors from patients with a variety of anemias (16). In a recent study (19) also observed an increase in the reaction

during

development

of eryth-

and polymorphonuclear leucocytes. all these studies the reaction product in the form of electron-dense particles

a diameter sponsible

of 10-60 nm for the brown-black

microscopy. Our results T. cruzi. The expected since

which stain

show that basic localization in the DNA-histone

possibly seen

apwith

is reby light

proteins exist in the nucleus was association has

been observed in almost all eucaryotic cells examined. In the kinetoplast the reaction depends on the developmental stage of the parasite. In epimastigotes, a stage in which the cells are able to divide, reaction was practically absent or weak. When present, particles were observed only

at the

periphery

in trypomastigotes ated with the

of the reaction

DNA

fibrils.

DNA was Since

disk. found very

However, associfew par-

of these of the

technique

particles

method.

(19) who granules

with

various

a special

cells. It has (15). PTA

proteins which reaction

cyleu-

anionic micros-

been widely is currently

modifications,

to

for

identify itself. It solution

polysaccharides

studies show it does not

(22).

that in some deterdetect polysacchaand Glick

histochemical (26) suggested

PTA selectively stains proteins. Many authors have treated cells,

glutaraldehyde, detect basic (5, 6) show

by

in the

PTA is an to electron

rides. On the basis of chemical experiments Silverman and that

arginine-rich described

proteins, or even DNA that PTA in aqueous affinity

However, other mined conditions

also

(14). They showed that it was for staining specific regions

of the fibrils of muscle used in negative staining polysaccharides, has been indicated

merely were

or in cytoplasmic in these cases the

acid: introduced

copy by Hall et al. particularly suitable

of the exclude

being

Particles

observed particles of polymorphonuclear

Phosphotungstic stain which was

has

periphery we can not

is detecting non-histone Similar results were

proteins.

used,

the

free in the cytoplasm It is possible that

observed vacuoles.

cocytes.

arginine-rich histones” (4). As pointed out by these authors “differences in AS staining may reflect differences in reactivity of amino and guanidino groups rather than differences in the relative content of lysine in the histone.” The AS technique was microscopy by MacRae

possibility

by

“yellow histones,

is associated

the

observed at of epimastigotes

contaminants

AS

and Ansley (3) and was used for investirelated with the biological functions of (3, 4, 21). It was shown in studies of the the

tides were kinetoplast

MacRae toplasmic

microscopy

AS staining of isolated histones that staining appears to indicate lysin-rich

DE SOUZA

prefixed

in

with PTA in absolute ethanol proteins. Bloom and Aghajanian that E-PTA binds principally

to to

rich in lysine, arginine, and histidine are localized at synapses. They also found in the nucleus. The same technique was

used by Sheridan nuclear proteins,

and Barrnett presumably

meiotic chromosomes lily microsporocytes.

of the Dense

(24) histones,

to

prophase material

locate in the

nuclei was

of ob-

served in the lateral element of the synaptinemal complex, in the chromatin, in the nucleoli, in material found in nuclear membrane pores and in the annulate lamellae. The results we obtained by applying E-PTA in T. cruzi support the view that this technique, in the conditions used here, did not detect carbohydrates.

Previous

studies

(10,

11)

the periodic acid-thiosemicarbazide-silver teinate technique was used (30) show cruzi, carbohydrates are associated the cell membrane or the membranes cytoplasmic react with

DNA

prothat in T. only with of some

vesicles. These membranes E-PTA. Dense material was

in the nucleus, at plast of epimastigotes of

in which

fibrils

trypomastigotes.

the

which

periphery of the and in the entire form

A reaction

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did not observed

the which

kinetobundle

kinetoplast appears

of in

BASIC some

other

structures

Some with

in the

localized

deserves consideration: pear to react with PTA in very proteins

PROTEINS

(a) which

thin sections. in ribosomes

cytoplasmic E-PTA. We

The can

cytoplasm

ribosomes apbe seen clearly

The presence has been reported

granules are not

(see sure

of

to have basic proteins. also react with E-PTA.

was seen flagellum,

especially reaction

peripheral pair

doublet

not

that they represent case they

sections. only

microtubules. did

react the

react

TRYPANOSOMA

used. The resolution

E-PTA technique since the silver

technique

have

have

only

microtubules A less intense

The

central

of the

trypanosomatids

suggest-

in the

morphology

in all staining

presence of Trypanosomatidae

proteins family.

dyes fast histones

in protozoa Steinert (28)

green and bromphenol by light microscopy

stream forms of Trypanosoma ture forms of Trypanosoma

mega

and a staining

Blastocrithidia of the nucleus

studied. However, the kinetoplast the DNA with basic Stewart taining

and Beck an antibody

detect histones Sarcomastigophora. linked species

histone tested

organelle Beck

panosomatids. Thivolet et al.

Few the of used

the the

blue to in bloodcul-

culicis. He observed of all trypanosomatids was observed suggesting

in 67 species of the They did not in any included

ponents Our

than data

post-formalin

electrophoresis, that

of calf

subphylum fmd DNA-

obtained gambiensi.

by

com-

thymus.

show similar results AS or the E-PTA

are

ki-

no alterations

kinetoplast

when

the

In T. cruzi, observed. Our

differentiation. can be easily E-PTA increase

The

of DNA in In most

or AS techniques in reaction of the when

fibrils

epimastigotes

trypomastigotes.

In

erythropoi-

etic cells, increase in the amount of silver granules was also observed when the maturation process was more advanced. There are at least two possibilities to explain the more intense reaction

T.

in the kinetoplast of trypomastigotes (a) during differentiation an increase

cruzi:

the

synthesis

of basic

differentiation

proteins

structural occur making for interaction

However,

a definitive

for will

need

previously with PTA answer,

to be carried

of in

(b) during of the

occurs;

rearrangements

proteins may sites available

masked or AS.

biochemical

out

to clarify

these

ACKNOWLEDGMENTS

The

the was

authors

are

Meyer

for

helpful

suggestions.

reading

most

grateful

to Dr.

Hertha

the

manuscript

and

giving

Mr.

A. L. de Oliv-

eira for help with the photography Sandra C. de Carvalho for secretarial

and Miss assistance.

We

thank

LITERATURE 1. Allen

RD,

Nomarski

David

tone 3. Black

in these MM,

ammoniacal 4. Black MM,

CITED

GB,

differential

transmitted-light 69:193, 1969 2. Beck JS, Walker nuclei: evidence

vealed

when either technique

into

cell.

in

serum conhistone to

more

there of the

DNA

transform

per

a condensation of the mitochondrion.

with either a considerable

that

However, biochemical analysis performed on Crithidia oncopelti (18) showed the presence of histone associated with the DNA of this trypanosomatid. The isolated histone contains, by polyacrylamide-gel

diameter.

and trypobasic proteins

problems.

of the 40 parasitic 28 species of try-

Similar results were (31) in Trypanosoma

represents portion

undergoes differences

studies

was not associated and Walker (2) and

(29) used human to DNA-linked

antigen which

porwas

lewisi, and cruzi, Trypanosoma

no staining (kinetonucleus)

of that proteins.

results show

pig sperm staining in

pair of microtubules. in trypanosomatids: published concerning

acid locate

mitochondrion

thus

seen on the central Basic proteins studies have been basic

one

netoplast a localized

kinetoplastic

the peripheral doublet tions of the flagellum.

larger

that epimastigote of T. cruzi have

In the at the

E-PTA flagellum.

guinea PTA

a better of the AS

associated with nuclear DNA. In the kinetoplast, basic proteins were found only at the periphery of the DNA rod of epimastigotes while they were

parasite however,

to study the found a dense

permits particles

a considerably

They also show mastigote forms

ing the existence of some differences between the two microtubules. We did not observe reaction on the filaments which form the para-flagellar structure. Gordon and Bensch (13) applied staining They

357

CRUZI

found in all DNA fibrils of the trypomastigote form. It is well established that the trypanosomatids

(c) Some microThis reaction

in very thin was observed

of microtubules

basic (8). (b)

Fig. 14) yet about

nature of these granules. It is possible correspond to lysosomes or that they a storage of metabolic products. In any appear tubules

IN

Nomarski interference

G:

microscopy. PJ: that

protozoa. Ansley

Z

Antigenicity DNA is not

Nature HR:

The

Zeiss-

equipment

Tech

of trypanosoma coupled to his-

204:194,

Histone

for

Mikrosk

1964

staining

with

silver. Science Ansley HR:

by ammoniacal

143:693, 1964 Histone specificity resilver staining. J Histochem

Cytochem 14:177, 1966 5. Bloom FE, Aghajanian GK: Cytochemistry

Downloaded from jhc.sagepub.com at IOWA TESTING PROGRAMS on March 16, 2015

of syn-

358

SOUTO-PADRON apses:

6.

selective

staining

for electron

Science 154:1575, 1966 Bloom FE, Aghajanian GK: cytochemical analysis of the junctions with phosphotungstic Res 22:361, 1968

7. Camargo panosoma somes Paulo

Fine staining acid.

media.

Rev

De

structural and of synaptic J Ultrastruct

Souza

W,

pomastigote

Chiari form

E: Fine of

from

acellular culture Bras Biol 37:671, 1977 De Sousa W, Grynberg Aspectos ultraestruturais

Inst

Biochem 19. MacRae proteins

Trop

of the tryisolated in column. Rev

cruzi

passage

21.

22.

F:

23.

do Trypanosoma cruzi em meio LIT. Rev Soc Bras Med Trop 9:143, 1975 11. De Sousa W, Meyer H: An electron microscopical and cytochemical study of the cell coat of Trypanosoma cruzi in tissue culture. Z Parasitenkd

24.

10.

46:179,

N, da

Nery-Guimar#{227}es forma epimastigota

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