ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 2227–2237 doi:10.1242/jcs.
135491
RESEARCH ARTICLE
The three-dimensional structure of the cytostome-cytopharynx
complex of Trypanosoma cruzi epimastigotes
Carolina L. Alcantara1,2, Juliana C. Vidal1,2, Wanderley de Souza1,2,3 and Narcisa L. Cunha-e-Silva1,2,*
ABSTRACT
The cytostome-cytopharynx complex is the main site of endocytosis
of Trypanosoma cruzi epimastigotes. Little is known about the
detailed morphology of this remarkable structure. We used serial
electron tomography and focused-ion-beam scanning electron
microscopy to reconstruct the entire complex, including the
surrounding cytoskeleton and vesicles. Focusing on cells that had
taken up gold-labeled tracers, we produced three-dimensional
snapshots of the process of endocytosis. The cytostome
cytoskeleton was composed of two microtubule sets – a triplet
that started underneath the cytostome membrane, and a quartet
that originated underneath the flagellar-pocket membrane and
followed the preoral ridge before reaching the cytopharynx. The
two sets accompanying the cytopharynx formed a ‘gutter’ and left a
microtubule-free side, where vesicles were found to be associated.
Cargo was unevenly distributed along the lumen of the cytopharynx,
forming clusters. The cytopharynx was slightly longer during the G2
phase of the cell cycle, although it did not reach the postnuclear
region owing to a bend in its path. Therefore, the cytopharynx is a
dynamic structure, undergoing remodeling that is likely associated
with endocytic activity and the preparation for cell division.
KEY WORDS: Cytostome, Trypanosoma cruzi, Endocytosis,
Electron tomography
INTRODUCTION
Endocytosis comprises a complex series of events that culminates
in the internalization of extracellular molecules and particles. This
process is well characterized in mammals and yeast, where
extracellular cargo enters the cell through invaginations of the
plasma membrane that are distributed all over the cell surface and
passes, in an orderly manner, through the different intracellular
compartments that characterize the endocytic pathways of these
organisms. In most protozoan parasites from the Trypanosomatidae
family, such as Trypanosoma brucei and Leishmania sp.,
endocytosis is highly polarized and restricted to the flagellar
pocket (Webster and Russell, 1993), an invagination of the plasma
membrane that surrounds the site at which the flagellum emerges
from the cell body. This restriction in the site of endocytosis is due
to the fact that the flagellar pocket membrane is not associated with
1
Instituto de Biofı́sica Carlos Chagas Filho, Universidade Federal do Rio de
Janeiro, Rio de Janeiro 21941-902, Brazil. 2, Instituto Nacional de Ciência e
Tecnologia em Biologia Estrutural e Bioimagens, Rio de Janeiro 21941-902, Brazil.
3
Diretoria de Metrologia Aplicada às Ciências da Vida, Instituto Nacional de
Metrologia, Qualidade e Tecnologia (Inmetro), Rio de Janeiro 20261-232, Brazil.
*Author for correspondence (narcisa@biof.ufrj.br)
Received 20 May 2013; Accepted 7 February 2014
the subpellicular microtubules that are positioned just beneath the
plasma membrane elsewhere in the cell (Angelopoulos, 1970),
preventing vesicular traffic.
This situation is different in Trypanosoma cruzi, the etiological
agent of Chagas disease. Epimastigotes of T. cruzi are the main
proliferative forms in the insect vector and have intense endocytic
activity, being able to endocytose different types of macromolecules
– such as transferrin, albumin, peroxidase and low-density
lipoprotein particles – from the extracellular milieu (Soares and de
Souza, 1991). Nevertheless, in this stage of the life cycle, the main
site of endocytosis is the cytostome-cytopharynx complex (Porto-
Carreiro et al., 2000), rather than the flagellar pocket. The
cytostome-cytopharynx complex consists of a deep invagination of
the plasma membrane, where the opening at the cell surface is
named the ‘cytostome’ and the long tube that forms the invagination
is called the ‘cytopharynx’. There is no defined limit between the
cytostome and the cytopharynx described so far. This structure is
also present in intracellular amastigote forms, although its endocytic
capacity has rarely been demonstrated (Meyer and de Souza, 1973).
During the differentiation of the epimastigote to the infective
trypomastigote form this complex disappears. Despite its importance
for the endocytic process, little is known about the structural
organization of the cytostome-cytopharynx complex. The shortage
of structural details make it difficult to understand how endocytosis
occurs through this structure. What is known is that there is a special
plasma membrane domain between the cytostome and the flagellar
pocket. This domain is characterized by a prominent glycocalyx and
is known as the ‘preoral ridge’ (Martı́nez-Palomo et al., 1976;
Pimenta et al., 1989; VataruNakamura et al., 2005). Also, an
uncharacterized set of microtubules and vesicles are aligned with the
cytopharynx (Milder and Deane, 1969), but the microtubule
arrangement and the origin and function of the aligned vesicles
are still unknown.
In this work, we elucidate the ultrastructure of the cytostome-
cytopharynx complex by using focused ion-beam-scanning
electron microscopy (FIB-SEM) and serial electron tomography,
followed by three-dimensional (3D) reconstruction. This was
coupled to the visualization of the endocytosis of labeled tracers, in
order to define the function of the different domains of the
cytostome-cytopharynx in the internalization of macromolecules.
The resulting detailed model of the entire cytostome-cytopharynx
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complex and its associated elements provides new insights into
how this structure performs the endocytic function in T. cruzi
epimastigotes.
RESULTS
Shape and length of the cytopharynx
Reconstructing a deep and curved invagination such as the
cytopharynx by serial ultrathin sections or even by serial electron
tomography is very labor intensive and is inappropriate for
statistical analyses, which require 3D reconstruction of a
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significant number of cells. By contrast, the powerful new

technique of FIB-SEM allows rapid 3D reconstruction of
organelles in a larger number of cells, albeit with less
resolution than electron tomography. It combines the serial
removal of material from the surface of a specimen (which
happens inside the microscope, using focused ion-beam milling)
with imaging of the newly exposed surface by scanning electron
microscopy (Soares Medeiros et al., 2012). We used FIB-SEM to
reconstruct the entire cytopharynx of epimastigotes that had taken
up horseradish peroxidase (HRP) for 5 minutes at 28 ˚C. The
choice of HRP as a tracer was based on our previous work (de
Souza et al., 1978; Soares and de Souza, 1991) and on the
assumption that, as a marker for fluid-phase endocytosis, it would
distribute homogeneously through the lumen of the cytopharynx,
thereby facilitating the recognition of this structure in FIB-SEM
images. The tracer proved to be suitable, as it filled only the
cytopharynx and made it prominent in all cells. However, we
observed sites of HRP concentration along the cytopharynx and a
considerable variation in the diameter of the cytopharynx along
its length (Fig. 1; supplementary material Movie 1).
Three slice-and-view series of FIB-SEM were used to measure
the length of the cytopharynx in 32 cells. The mean cytopharynx
length was ,8 mm (Fig. 2A). In these samples, there was a
mixture of cells in different stages of cell cycle; thus, we
classified these cells based on their stage of cell cycle, assuming
that epimastigotes with one nucleus, one kinetoplast and one
flagellum (1N1K1F) were in G1, whereas epimastigotes with one
nucleus, one kinetoplast and two flagella (1N1K2F) were in an
early stage of G2 (Elias et al., 2007). We noticed a small, albeit
significant, increase in the length of the cytopharynx in parasites
in G2, compared with those in G1. (Fig. 2A). The mean
cytopharynx length of cells in G1 was 6.8 mm61.9 mm (n516;
6s.d.), whereas cells in G2 had a mean cytopharynx length of
8.9 mm61.4 mm (n516). Moreover, during G1 the cytopharynx
had a helical shape, bypassing the nucleus and reaching the
posterior of the cell (Fig. 2B–D), whereas in G2 there was a bend
in the path of the cytopharynx, at a region close to the anterior
portion of the nucleus, that turned the cytopharynx towards the
anterior region of the cell (Fig. 2E–G). As a result of this bend,
the cytopharynx of G2 parasites did not reach the posterior of the
cell.

The ultrastructural organization of the cytostome-

cytopharynx complex
To understand the ultrastructural organization of the cytostome-
cytopharynx complex we used high resolution electron
tomography. Owing to the long length of the complex, we
analyzed several tomograms of different parts of the complex,
taken from different cells. To preserve the architecture of the
complex, after cells had taken up the endocytic tracer [transferrin
coupled to colloidal gold (Tf–Au)] for 10 minutes, we added
isothermal glutaraldehyde directly to the incubation medium
Fig. 1. Variation in diameter and HRP accumulation along the length of
the cytopharynx. Parasites that had taken up HRP for 5 minutes were
imaged using FIB-SEM. (A–F) Sequence of images of a cell showing
different portions of the cytostome-cytopharynx complex (white arrows 1–6),
from anterior to posterior. (A) The cytostome (arrowhead) at the anterior of
the cell was devoid of HRP. (B) Towards the posterior of the cell, a
transversal section of the cytopharynx displayed an empty lumen and a small
diameter (arrow 1). (C) A longitudinal section of the cytopharynx showing an
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(Gadelha et al., 2009), to increase the speed of fixation and
minimize cytoskeleton and membrane destabilization.
The first tomogram (Tomo 1, Fig. 3; supplementary material
Movie 2) was produced by joining tomograms of seven adjacent
200-nm sections, resulting in 597 virtual slices, each 1.5-nm
thick. The reconstructed volume comprised part of the flagellar
pocket and the region of the cytostome opening. In this
transversely positioned epimastigote it was possible to see the
entire preoral-ridge membrane domain, characterized by an
electron-dense surface coat running from inside the flagellar
HRP-filled lumen and two regions with different diameters (arrows 2 and 3).
(D,E) Transversal sections showing the HRP-filled distal portion of the
cytopharynx, highlighting once more the uneven diameter of this structure
along its length (arrows 4 and 5, respectively). (F) Distal end of the
cytopharynx (arrow 6) at the posterior of the cell. (G) A 3D model of the cell
shown in A–F. The respective regions of the cytopharynx indicated in A–F
are represented in the model (arrows 1–6). R, reservosomes (red, the
lysosome-related organelles of T.cruzi epimastigotes); N, nucleus (blue); K,
kinetoplast (green); G, Golgi complex; M, mitochondrion; F, flagellum
(orange); FP, flagellar pocket (white). Scale bars: 1 mm. The complete
imaging, by FIB-SEM, of the cell shown here can be found in supplementary
material Movie 1.

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pocket (Fig. 3A–D, purple brackets) to a position close to the
cytostome (Fig. 3C,D, purple brackets; Fig. 3E,F, purple
surface). In the cytoplasm, a set of four microtubules was
observed underlying this membrane domain (Fig. 3A–D), then
lining the cytopharynx and going deep inside the cell body
(Fig. 3G–I). A different set of three microtubules initiated just
below the cytostome membrane and also accompanied the
cytopharynx (Fig. 3B,G,I). These two sets of microtubules were
arranged in a ‘gutter’ shape around the cytopharynx, leaving a
side of the cytopharynx membrane devoid of underlying
microtubules (Fig. 3I).
We confirmed these findings by analyzing a second tomogram
of the same region in a different cell (Tomo 2; 321 virtual slices,
each 3.14-nm thick). In this tomogram (Fig. 4), we could clearly
observe the existence of the four microtubules that have one end
underneath the flagellar-pocket membrane and follow the path of
the preoral ridge (Fig. 4A). We noticed that, as in Tomo 1
(Fig. 3G), the flagellar-pocket end of these microtubules was not
found in the same transversal plane relative to the flagellar-pocket
opening. We numbered the microtubules from the quartet from
Q1 to Q4. As seen in the model generated from Tomo 2
(Fig. 4A), microtubules Q1 and Q2 from the quartet were already
present in the last slice of the tomogram (z-slice number 321). By
contrast, microtubules Q3 and Q4 appeared at virtual slices 237
and 205, respectively. Therefore, the flagellar-pocket end of these
microtubules was found at a more anterior position, closer to the
Journal of Cell Science (2014) 127, 2227–2237 doi:10.1242/jcs.135491
Fig. 2. Analysis of cytopharynx
length and shape. By using FIB-SEM
microscopy we were able to reconstruct
and measure the length of the
cytostome-cytopharynx complex in 32
cells that had endocytosed HRP for
5 minutes. (A) Morphometric analysis of
the length of the cytopharynx of T. cruzi
epimastigotes in all cells analyzed (Total)
and in cells from G1 and early G2 phase
of the cell cycle. The mean cytopharynx
length when all cells were analyzed was
8.0 mm62.0 mm (6s.d.). The mean
cytopharynx length of cells in G1 was
6.8 mm61.9 mm, whereas cells in G2
had a mean cytopharynx length of
8.9 mm61.4 mm. The data was analyzed
using an unpaired t-test and post-
analyzed by two-tailed test. *P,0.01.
(B–D) 3D models of the cytopharynx (Cy,
pink) in three representative G1-phase
cells. The cytopharynx followed a helical
path around the nucleus (N, blue),
reaching the posterior of the cell. White
arrowhead, the cytostome. (E–G) 3D
models of the cytopharynx in G2-phase
cells showing a bend in this structure (*)
towards the anterior of the cell. K,
kinetoplast (green). Scale bars: 0.5 mm.
opening of the flagellar pocket (Fig. 4A). The microtubule triplet
was first observed in this tomogram close to the cytostome
opening (Fig. 4A), and its microtubules were numbered T1 to T3,
with T3 being the closest to microtubule Q4 from the quartet
(Fig. 4D–F). Interestingly, some of these microtubules
disappeared as the cytopharynx became thinner, projecting
towards the interior of the cell (Fig. 4E,F). The first to
disappear was microtubule Q3 (Fig. 4E), followed by T1
(Fig. 4F), leaving five microtubules around the cytopharynx on
one end of Tomo 2 (Fig. 4F,I). In this tomogram, an endocytic
vesicle containing Tf–Au particles was observed very close,
although not fused, to the ‘naked’ side of the cytopharynx (i.e. the
side where the cytopharynx membrane is devoid of associated
microtubules) (Fig. 4D,G,H). In the cytoplasm, we could find
other membranous elements in close proximity to the
cytopharynx (Fig. 4E,F), although we could not unambiguously
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identify them, because they were on the edge of the tomogram.
Internalization of the endocytic tracer Tf–Au occurred by
vesicle budding along the ‘naked’ side of the cytopharynx
The arrangement of the microtubules along the cytopharynx has
an important consequence for the architecture of the endocytic
process that takes place in the cytopharynx. This could be best
illustrated in Tomo 3 (Fig. 5), which covers 3.62 mm in length of
the cytopharynx, with a slice thickness of 3.14 nm. Fig. 5A gives
an overall view of the model generated from the reconstructed
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Fig. 3. Tomo 1, the 3D organization of the cytostome-cytopharynx complex and its association with the cytoskeleton. Serial tomography of an
epimastigote that had endocytosed Tf–Au for 10 minutes. (A–D) Set of four z-slices going from posterior to anterior regions of the cell, showing the preoral ridge
(purple brackets) extending from the flagellar pocket (FP) to the cytostome (Cy), and the underlying microtubule quartet (blue arrows), which appears to give
mechanical support to the cytostome-cytopharynx together with a microtubule triplet (green arrows in B). F, flagellum; M, mitochondrion. (E) Model of
Tomo1 showing the flagellar-pocket region and the emergence of the preoral ridge (purple) from the flagellar pocket (rectangle, white arrow). (F) The
same model observed from a different position, showing the cytostome (pink, white arrowhead) and the presence of Tf–Au (dots) adhered to the surface of
the preoral ridge. (G,H) Cytoplasmic views of the model. The microtubule quartet (blue) and the flagellar pocket (white) edge of the preoral ridge (purple arrow)
can be seen, along with the microtubule triplet (green) that initiates at the base of cytostome. In I, the microtubule triplet and quartet can be seen around
the cytostome opening and following the path of the cytopharynx, leaving a microtubule-free ‘naked’ side of the cytostome-cytopharynx membrane (black
arrowhead). Scale bars: 200 nm.

tomogram. Many uncoated vesicles, loaded (light blue vesicles)
or not (silver vesicles) with Tf–Au particles, could be seen to be
aligned with the naked side of the cytopharynx, along the length
of this structure (Fig. 5A,B). We noticed that the Golgi complex
was in close proximity to the cytopharynx (Fig. 5B). Importantly,
vesicles containing Tf–Au (Fig. 5E,F) or without this tracer
(Fig. 5C,D) were observed clearly budding from or fusing with
the cytopharynx membrane. All vesicles aligned with the
cytopharynx had the approximate diameter of 90 nm, and the
gold-containing vesicles seemed to be more frequently found at
could be observed and should correspond to the early endosomes
of this organism (Fig. 5G).
Fig. 6 shows a serial tomogram that covers the final portion of
the cytopharynx (Tomo 4; 239 virtual slices, each 1.9 nm thick).
We could find Tf–Au particles gathered in clusters inside
enlarged portions of the cytopharynx, separated by regions
devoid of the tracer (Fig. 6A,B), as we observed in cells that had
taken up HRP. Therefore, we have shown that, when it is engaged
in endocytosis, the cytopharynx varies in diameter along its
length. Although the variations in diameter and length of the
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the distal portion of the cytopharynx.
Tf–Au particles were not evenly distributed inside
the cytopharynx
The distribution of Tf–Au particles inside the cytopharynx was
not homogeneous, forming expansions where the tracer
concentrated. In Fig. 5A,G, a dilated portion of the cytopharynx
was sectioned at the edge of the tomogram. Close to this dilated
portion, endocytic compartments with a tubule-vesicular
morphology and containing a large number of gold particles
cytopharynx have been observed using different techniques,
different tracers and in different cells, we cannot rule out some
contribution of osmotic variation inherent to chemical fixation.
The cytopharynx ended in a large bag-shaped structure,
positioned very close to the reservosomes (a lysosome-related
organelle of T. cruzi epimastigotes) at the posterior of the cell
(Fig. 6C). At this point, only four microtubules (two from each
set) were observed around the cytopharynx, and they seemed to
continue even after the cytopharynx ended (Fig. 6B,C). In all
cells we have examined so far, after 2–10 minutes of incubation

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Fig. 4. Tomo 2, the distribution of microtubules along the cytopharynx. Serial tomograms of an early-G2-phase (1N1K2F) epimastigote that had taken up
Tf–Au for 15 minutes, showing the positioning of microtubules along the cytopharynx. (A) View of the 3D model of Tomo 2, showing the beginning of the
microtubule quartet (Q1 to Q4, blue) that follows the path of the preoral ridge (purple) and then bends into the cytoplasm, accompanying the cytostome-
cytopharynx (pink). A microtubule triplet (T1 to T3, green) originates from a position adjacent to the opening of the cytostome and also accompanies the
cytopharynx. (B–F) Virtual slices of the tomogram representing the regions indicated in A. (B) The microtubule quartet (blue arrows) can be seen lining the
cytopharynx (Cy) and the preoral ridge (purple contour). In D we can see the full set of microtubules around the cytopharynx and a vesicle (black arrowhead)
containing Tf–Au particles (black dots) close to the naked side of the cytopharynx. This vesicle can also be seen in yellow in the model in G and H. Other
membrane-bound compartments were observed close to the naked side of the cytopharynx (asterisks in E and F). As the cytopharynx becomes thinner, running
deeper into the cell, microtubules Q3 from the quartet (E) and T1 from the triplet (F) disappear. The light blue arrow and the dark green arrow in E and F,
respectively, indicate the very end of these microtubules. (G) Back view of the model in A, in which the positioning of microtubules at the flagellar pocket,
preoral ridge and the cytostome can be better visualized. (I) At the most distal portion of the cytopharynx (relative to the cytostome) only five microtubules were
seen around this structure, arranged in a half-moon shape. K, kinetoplast; M, mitochondrion; G, Golgi complex; F, flagellum ; orange, new flagellum; light blue,
old flagellum. Scale bars: 200 nm

with endocytic tracers, the tracers were contained in the
cytopharynx, which ended in a bag-shaped dilation.
The flagellar pocket of T. cruzi epimastigotes has two
The MtQ underlies a membrane domain in the flagellar pocket
that forms a channel through which extracellular components
can gain access to the lumen of the flagellar pocket (Gadelha
et al., 2009).
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microtubule quartets We performed a serial tomogram of a cell that covered the

The existence of a microtubule quartet that is common to both
the flagellar pocket and the cytostome-cytopharynx complex led
us to question whether these microtubules correspond to a
conserved quartet of microtubules that surrounds the flagellar
pocket in other trypanosomes (Preston, 1969; Taylor and
Godfrey, 1969; Lacomble et al., 2009; Girard-Dias et al.,
2012). In T. brucei, this conserved quartet is often referred to as
the MtQ (microtubule quartet), and it comprises the only
microtubules associated with the flagellar-pocket membrane.
entire volume of the flagellar pocket (Tomo 5, Fig. 7). The
reconstructed volume, consisting of 771 virtual slices, each 1.58-
nm thick (Fig. 7E), demonstrated the presence of two different
quartets of microtubules underlying the flagellar-pocket
membrane of T. cruzi epimastigotes. One of the quartets
originated close to the basal body, surrounded the flagellar
pocket in a helical pattern and inserted into the subpellicular
microtubule cortex, near the opening of the flagellar pocket
(Fig. 7A–E). This clearly corresponded to the MtQ.

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The microtubules of the other quartet represented the ones
shown in Tomos 1 and 2. One end of these microtubules was
positioned below the flagellar-pocket membrane, but anterior to
the basal body area. Also, the end of each microtubule was found
at a different position relative to the anteroposterior axis of the
cell (Fig. 7A–C,E). Their path accompanied that of the preoral
ridge, and then these microtubules extended into the parasite
body, along with the cytopharynx. To investigate these
microtubules in the context of the entire cytoskeleton, we
detergent-extracted the membranes of epimastigotes and
negatively stained the resulting whole-mount cytoskeletons for
observation by transmission electron microscopy (TEM). In these
preparations, we could identify the two different microtubule
quartets and follow the cytostome quartet from the flagellar
pocket to the posterior region of the cell (Fig. 8A–C).
Interestingly, the arched shape of the cytostome microtubules
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Fig. 5. Tomo 3, the active membrane
domain of the cytopharynx.
Tomographic reconstruction of the distal
portion of the cytopharynx of an
epimastigote that had taken up Tf–Au for
15 minutes. (A) The model represents
the cytopharynx (pink) with its associated
microtubules (green and blue) and a
series of vesicles (light blue and silver)
adjacent to the cytopharynx. A portion of
the Golgi complex (red) was also
modeled to show its proximity to the
cytopharynx. (B) Some of the vesicles
aligned to the cytopharynx contained Tf–
Au (light blue vesicles), whereas others
were empty (silver vesicles). (C,D) In C, a
virtual image of the tomogram shows an
empty vesicle (arrow) in direct contact
with the cytopharynx membrane. This is
represented in the 3D model in D. (E,F) A
vesicle loaded with Tf–Au can also be
seen in direct contact (i.e. undergoing
fusion or fission) with the cytopharynx
membrane (arrow). (G) The cytopharynx
(Cy) presents a dilated distal portion filled
with Tf–Au. A series of tubule-vesicular
compartments, probably the early
endosomes, loaded with Tf–Au (yellow)
are seen close to the cytopharynx.
Orange, flagellum. Scale bars: 200 nm
(A–D,G); 100 nm (E).
as they pass underneath the predicted region of the preoral ridge
was always preserved in the cytoskeletons, even after total
membrane extraction (Fig. 8A,B). This region appeared to be
structurally complex relative to other areas of the cytostome-
cytopharynx cytoskeleton, where microtubules appeared
comparatively ‘bare’ (in Fig. 8, compare the structures of the
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cytostome-cytopharynx cytoskeleton in B and C). The
cytopharynx followed a helical path around the nucleus from
anterior to posterior. Also, our negative-staining images
confirmed that the anterior portion of the cytopharynx
microtubules always reached the plane of the nucleus from the
opposite side of the cell relative to the one where the cytostome is
found. These microtubules remained closely associated with each
other along the entire path to the posterior of the cell, even
without the cytopharynx membrane. We could count five
cytopharynx microtubules immediately posterior the nucleus,
RESEARCH ARTICLE Journal of Cell Science (2014) 127, 2227–2237 doi:10.1242/jcs.135491

Fig. 6. Tomo 4, Tf–Au particles are found in clusters in the lumen of the cytopharynx. (A) Projection of all images from the tomogram onto a single
plane, rotated so that the final portion of the cytopharynx (Cy) can be seen almost in its entirety. White arrows, regions of concentration of Tf–Au. (B) A
model of Tomo 5 showing the regions of enlarged diameter in the cytopharynx (pink), where Tf-Au is concentrated (white arrows). Vesicles (light blue)
can also be observed close to the cytopharynx. Note that the remaining cytopharynx microtubules extend beyond the end of cytopharynx (arrowhead). (C) Top
view of the model showing the distribution of microtubules around the cytopharynx. Only four microtubules (two of each set) can be observed around the
distal portion of the cytopharynx (inset). Note the proximity between the end of the cytopharynx and the reservosomes (R, blue contour). Scale bars:
200 nm.

and these were reduced to four towards the end of the
cytopharynx (Fig. 8A–C).
In T. brucei, the subpellicular microtubule corset is known
to depolymerize after treatment with CaCl2 or high concentrations
of salt, whereas the flagellar microtubules and the MtQ remain
(Gull, 1999). We confirmed that this was also the case in
T. cruzi epimastigotes by treating cytoskeletons with 1 M NaCl
(Fig. 8D) or 60 mM CaCl2 (Fig. 8E). Importantly, the cytostome
cytoskeleton was also resistant to these salt treatments. Distal to the
cytostome region, sections of varying lengths of the cytopharynx
microtubules remained in salt-treated cytoskeletons (not shown).
As expected, the subpellicular microtubules disappeared upon salt
treatment.
In our reconstruction of the flagellar pocket (Tomo 5), we have
also found other recently described cytoplasmic microtubules
(Girard-Dias et al., 2012). These were not included in the model
shown in Fig. 7, because they are not related to the cytostome-
cytopharynx microtubules. All of the microtubules we could
observe near the flagellar pocket in Tomo 5 are shown in
supplementary material Fig. S1.
In Tomo 5, two rare invaginations were observed at the
flagellar-pocket membrane; one of them appeared to represent a
small budding vesicle of 32 nm in diameter (supplementary
material Fig. S2A,B; Box 1), and the other resembled a precursor
of a tubule-vesicular structure (supplementary material Fig.
S2A,C; Box 2). A large vesicle, measuring 274 nm in diameter
(supplementary material Fig. S2D,E; Box 3), was also seen in
direct contact with the flagellar-pocket membrane. Note that,
two-dimensional (2D) micrographs. In the present work, we
used high resolution electron tomography combined with FIB-
SEM microscopy to generate the first integrated view of the 3D
architecture of the cytostome-cytopharynx complex in T. cruzi
epimastigotes.
Several characteristics of this structure could be addressed. The
cytopharynx is a deep and spiral-shaped invagination, with
considerable variation in length from cell to cell. Parasites with a
shorter cytopharynx would present a decrease in endocytic
activity, as suggested by previous work that reconstructed one
epimastigote in the early G2 phase of the cell cycle using
conventional ultrathin serial sections (Ramos et al., 2011). In the
present work, using FIB-SEM to obtain 3D models and
quantitative information about the cytopharynx in several cells,
we confirmed the apparent retraction of the cytopharynx in G2,
but demonstrated that this was not due to shortening in length.
Rather, the cytopharynx become slightly longer in G2, but
appeared retracted owing to a change in its shape. Moreover, by
using the endocytic tracer, we could guarantee that all of the cells
in which the cytopharynx was measured presented endocytic
activity. Thus, cytopharynx remodeling from G1 to G2 was not
associated with an obvious decrease in the endocytic capacity of
the cells. Based on this, we strongly suggest that the changes in
cytopharynx shape and length in G2 reflect the remodeling of the
cytopharynx in preparation for cell division. The events involved
in the duplication of the cytostome-cytopharynx complex during
cell division are now under detailed investigation.
The participation of microtubules in the structuring of
Journal of Cell Science

although the endocytic tracer was present in the extracellular
milieu, no tracer was found inside the flagellar-pocket vesicles.
DISCUSSION
Since the initial description of the cytostome-cytopharynx
complex of T. cruzi epimastigotes (Milder and Deane, 1969),
very little further knowledge about this structure was gained,
despite its great importance as the main site of endocytosis at this
stage of the life cycle. This was due to the difficulty in following
this structure in its entirety when using only analyses of
the cytostome-cytopharynx complex is a hallmark of
trypanosomatids (Steinert and Novikoff, 1960; Preston, 1969)
and bodonids (Attias et al., 1996). However, the origin of these
microtubules and their exact positioning and arrangement around
the cytopharynx remained unclear. The existence of a physical
cytoskeleton-mediated link between the flagellar pocket and the
cytostome had been suggested by Okuda and co-workers (Okuda
et al., 1999). Nevertheless, a clear description of the structure of
this physical link was lacking, requiring the analysis presented
here using serial electron tomography of epimastigotes, where

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Fig. 7. Tomo 5, the microtubule cytoskeleton of the epimastigote flagellar pocket. The reconstructed model covers the entire volume of the flagellar-pocket
(FP) region of an epimastigote and includes part of the cytostome-cytopharynx (Cy). (A–D) Different virtual planes of the tomogram. (A) A transversal
image of the flagellar-pocket region (z-slice #332, indicated in the model in E), showing the microtubules Q1 and Q2 (blue arrows) of the cytostome-cytopharynx
quartet and the conserved quartet MtQ (orange arrows). (B,C) More-posterior images of the flagellar-pocket area (z-slices #242 and #181, also indicated in E),
showing the MtQ and the complete quartet of microtubules of the cytostome-cytopharynx underlying the preoral ridge (POR) domain and reaching the
cytostome. (D) In z-slice #16, the MtQ is seen under the plasma membrane region opposed to the flagellar membrane. (E) 3D model of the tomogram showing
the features highlighted in images A–D. The epimastigote flagellar pocket possesses two microtubule quartets. One of these quartets (MtQ, orange tubes)
originates between the basal body (BB, beige tubes) and the pro-basal body (PBB, yellow tubes) and surrounds the flagellar pocket (white) before becoming
inserted between the subpellicular microtubules (SPMT, white lines). Each microtubule of the second quartet (Q1–Q4, blue tubes) initiates individually at a
specific transversal plane relative to the flagellar-pocket opening (blue arrows). These microtubules pass just beneath the differentiated membrane domain of the
preoral ridge (purple) and follow the path of the cytopharynx (pink), together with the microtubule triplet (green tubes). A vesicle (yellow) can be seen close to the
flagellar pocket and is better described in supplementary material Fig. S2. F, flagellum (orange surface); K, kinetoplast; N, nucleus; beige surface, plasma
membrane. Scale bars: 200 nm

microtubules could be individually followed from the flagellar cytoskeletons, the cytostome-cytopharynx microtubule quartet
Journal of Cell Science

pocket all the way to the end of the cytopharynx by high-
resolution 3D reconstruction.
Our results describe the existence of seven microtubules
following the path of the cytopharynx of T. cruzi epimastigotes.
The new microtubule quartet described here follows the path of
the preoral ridge and of the cytopharynx. Therefore, this new
quartet is likely to be responsible for the arched shape of the
preoral ridge, and for the formation and/or maintenance of the
preoral-ridge membrane domain. Indeed, even after the
membranes were extracted for examination of whole-mount
remained together and described a helical path toward the
posterior of the cell, a strong indication that these microtubules
are responsible for the curved shape of the cytopharynx. The
reconstruction of the cytopharynx of several parasites from FIB-
SEM images confirmed this shape. It is possible that this is a
consequence of the positioning of the kinetoplast between the
cytostome and the nucleus, which might force the cytostome to
adopt a helical path towards the opposite side of the cell (as
opposed to running straight towards the posterior of the cell). We
could not identify clearly the cytostome portion of the

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microtubule triplet in whole-mount cytoskeletons. However, in
the perinuclear region, at least five microtubules could be counted
in the cytopharynx cytoskeleton. Therefore, at least one of these
microtubules must be part of the cytopharynx microtubule triplet
seen in tomographic reconstructions. It is possible that, in
negatively stained cytoskeletons, the cytostome end of the triplet
microtubules is obscured by the complexity of insoluble (i.e.
detergent-resistant) structures found in the region of the
cytostome. We speculate that the structural complexity of this
region is due to the presence of detergent-resistant components of
the preoral-ridge membrane domain that are stably linked to the
cytostome-cytopharynx microtubules.
The presence of a cortex of closely spaced subpellicular
microtubules beneath the epimastigote plasma membrane impairs
the events of budding or fusion of vesicles. Although the
cytostome-cytopharynx complex lacks microtubules of the
subpellicular cortex, it is nevertheless associated with two sets
of microtubules. Given that this complex is the main site of
endocytosis in epimastigotes, understanding the positioning of
microtubules around the cytostome-cytopharynx allowed us to
make important conclusions regarding the architecture of the
endocytic processes in this parasite. If the cytostome-cytopharynx
Journal of Cell Science (2014) 127, 2227–2237 doi:10.1242/jcs.135491
Fig. 8. Whole-mount negatively stained
cytoskeleton after detergent extraction.
(A) Detergent-extracted whole-mount cytoskeletons
of T.cruzi epimastigotes. (B) High magnification of the
area in rectangle B in A, showing the flagellar-pocket
region. The flagellar-pocket quartet (MtQ, orange
bracket) and the cytostome quartet (blue asterisks)
are indicated, as well as the predicted region for the
cytostome (Cy, white arrowhead). (C) High
magnification of the area in rectangle C in A, showing
the distal portion of the microtubules that accompany
the cytopharynx. In a more anterior region, five
microtubules (white asterisks) were seen, whereas at
a more posterior region only four were found.
(D,E) Resistant microtubule preparations were
obtained after treatment of the cytoskeletons with 1 M
NaCl (D) or 60 mM CaCl2 (E). In both cases, the
axoneme, the MtQ and the cytostome microtubule
quartet were preserved. N, nucleus; Axo, axoneme;
BB, basal body; PBB, probasal body. Scale bars:
500 nm (A–C,E), 200 nm (D).
microtubules formed a closed cage around all sides of the
cytopharynx, it is likely that the endocytosed cargo would have to
travel all the way to the bottom of the cytopharynx in order to be
endocytosed in budding vesicles. Instead, our tomograms showed
that these microtubules are arranged around the cytopharynx in a
‘gutter’ shape, leaving a side of the cytopharynx membrane
devoid of underlying microtubules. Close to this ‘naked’ side of
the cytopharynx, many uncoated vesicles could be observed. In
fact, some of these vesicles were connected with the cytopharynx
membrane and contained the endocytic tracer Tf–Au. Although
the presence of vesicles near the side of the cytopharynx is
documented in the literature (Milder and Deane, 1969; de Souza
Journal of Cell Science
et al., 1978), the occurrence of endocytosis in this region had
never been demonstrated. Therefore, by using a labeled endocytic
tracer, we have demonstrated that there is active membrane traffic
at the naked side of the cytopharynx. With the techniques used
here, we could not be certain whether the vesicles devoid of Tf–
Au that were observed in contact with the cytopharynx membrane
were budding from or fusing with this structure. We have shown
that the Golgi apparatus is in close proximity to the cytopharynx.
Thus, it is possible that the vesicles devoid of the gold tracer near
the cytopharynx originated from the Golgi complex. In this
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context, vesicles coming from the Golgi would deliver their
contents to the cytopharynx, maintaining and replacing its
differentiated membrane domain and acidifying its lumen.
Indeed, we have demonstrated that the lumen of the
cytopharynx is acidified by the action of a P-type H+-ATPase
(Porto-Carreiro et al., 2000; Vieira et al., 2005).
Some of the microtubules around the cytopharynx terminate as
this structure becomes thinner and goes deeper inside the cell.
Therefore, fewer microtubules are found at the end of the
cytopharynx, which is likely to contribute to the fact that this is
the preferred site of exchange of content with other compartments.
We have noticed that other endocytic compartments are often in
close proximity to regions of the cytopharynx where only four
microtubules are found. At these locations, the cytopharynx was
often enlarged and contained the endocytic tracer. Thus, the
diameter of the cytopharynx varies along its length. Until now, the
cytopharynx was described as having a funnel-shaped opening –
the cytostome – followed by a tube-like element with a roughly
homogeneous diameter along its length – the cytopharynx proper –
which terminates in a bag-shaped structure (Milder and Deane,
1969; de Souza et al., 1978). Based on this description, it was
assumed that endocytosis occurred exclusively at the enlarged end
of the cytopharynx. By contrast, our analyses of different serial
tomograms and FIB-SEM series showed that the cytopharynx
varies in diameter along its length. Also, the regions of enlarged
diameter along the cytopharynx contained clusters of endocytic
tracers, strongly suggesting that they are engaged in endocytosis.
This might have been missed in previous publications where only
2D images of the cytopharynx, from ultrathin TEM sections, were
analyzed. Depending on the plane of observation, an enlargement
of the cytopharynx along its length might have been mistaken for
the distal end of this structure, thus leading to the assumption that
endocytosis occurs exclusively at the end of the cytopharynx.
Interestingly, the aggregation of cargo in clusters along the
cytopharynx was observed when using Tf–Au, an insoluble
particulate tracer, as well as with peroxidase, a fluid-phase tracer,
showing that this feature is independent of the nature of the cargo.
The region where these clusters form along the cytopharynx
might characterize preferential ‘exit points’ where vesicles bud
into the cytoplasm. At the final portion of the cytopharynx, four
microtubules were still present and continued towards the
posterior of the cell, running past the end of the cytopharynx.
Although we did not observe any internal cell compartments
directly connected with these microtubules, one could speculate
that they play a role in directing organelles or vesicles towards the
posterior of the cell.
In conclusion, the 3D reconstruction work presented here has
proven to be more adequate for the analysis of the spatial
dynamics of endocytosis in epimastigotes, compared with
conventional methods of observation that have been used so
far. The high-resolution 3D analyses revealed new features of the
long and complex structure of the cytostome-cytopharynx and
opened the possibility of understanding the highly efficient
endocytic process in this interesting and relevant eukaryotic
model.
MATERIALS AND METHODS
Parasites
T. cruzi epimastigotes from clone Dm28c were cultivated in liver
infusion tryptose (LIT) medium (Camargo, 1964) supplemented with
10% (v/v) fetal calf serum (FCS) at 28 ˚C. Exponential-phase parasites
from 3–5-day-old cultures were used for the experiments.
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Journal of Cell Science (2014) 127, 2227–2237 doi:10.1242/jcs.135491
Endocytic tracers
Colloidal gold particles (Au, 8–10 nm) were prepared as described
previously (Slot and Geuze, 1985). Bovine holotransferrin (Tf, Sigma, St
Louis, MO) was coupled to gold particles as described previously (Roth,
1983). Horseradish peroxidase (HRP, Sigma) was used as fluid-phase
tracer at a final concentration of 1 mg/ml in culture medium.
Endocytosis experiments
Parasites were collected by centrifugation at 1500 g for 10 minutes and
were washed twice in phosphate-buffered saline [10 mM sodium
phosphate buffer with 0.9% (w/v) NaCl, pH 7.2]. Cells were incubated
in RPMI medium (without FCS) that contained transferrin coupled to
gold particles (Tf–Au) for 10 or 15 minutes; or HRP for 5 minutes, both
at 28 ˚C. To stop endocytosis, cells were fixed rapidly by the addition of
isothermal glutaraldehyde to a final concentration of 2.5% directly to the
incubation medium. After fixation, the cells were washed twice in PBS to
remove the endocytic tracer and processed for transmission electron
microscopy (TEM).
3-39-diaminobenzidine (DAB)-cytochemistry
Before processing for TEM, cells that had taken up HRP were incubated
in a solution containing 0.5 mg/ml DAB (Sigma) in Tris-HCl buffer,
pH 7.6, for 15 minutes in the dark and at room temperature. After this
incubation, H2O2 was added to a final concentration of 0.03% (v/v), and
the cells were kept in the dark for a further 15 minutes at room
temperature (Graham and Karnovsky, 1966). Cells were then washed and
processed for TEM.
Transmission electron microscopy
Cells were fixed by using 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate
buffer, pH 7.2, for 1 hour at room temperature, or fixed in isothermal
glutaraldehyde (after endocytosis, as described above). Following a
wash in cacodylate buffer, the cells were post-fixed using an osmium-
thiocarbohydrazide-osmium (OTO) protocol (Willingham and Rutherford,
1984). Briefly, the cells were incubated in a post-fixative osmium solution
[1% (v/v) osmium tetroxide, 0.8% (v/v) potassium ferrocyanide and 5 mM
calcium chloride in 0.1 M cacodylate buffer, pH 7.2] for 40 minutes,
washed twice in water and then incubated in a solution of 1% (w/v)
thiocarbohydrazide (TCH, Sigma) in water for 5 minutes. After three
washes in water, the cells were incubated again in the post-fixative osmium
solution for 3 minutes. Following post-fixation, the samples were washed in
water, dehydrated in an acetone series and embedded in epoxy resin.
Ultrathin sections were stained post-embedding with 5% (w/v) uranyl
acetate and lead citrate, and were observed by using a Tecnai-G2 electron
microscope operating at 200 kV.
Preparation of whole-mount cytoskeletons
Epimastigotes were mounted onto glow-discharged formvar-coated grids,
extracted with 1% (v/v) NP-40 in PEME buffer (100 mM PIPES, 1 mM
MgSO4, 0.1 mM EDTA and 2 mM EGTA, pH 6.9), followed by fixation
with 2.5% (v/v) glutaraldehyde in PEME (Parussini et al., 2003). For the
preparation of salt-treated cytoskeletons, grids were incubated in 1 M NaCl
or 60 mM CaCl2 immediately after detergent extraction and prior to fixation.
Grids were negatively stained with 0.7% (v/v) aurothioglucose in water and
observed using a Tecnai-G2 electron microscope operating at 200 kV.
Electron tomography
Journal of Cell Science
For electron tomography, ribbons of 200-nm thick serial sections were
produced from TEM blocks prepared as described above. These ribbons
were collected onto formvar-coated copper slot grids and stained post-
embedding as described above. Colloidal gold particles (10 nm) were
deposited on both surfaces of the sections, to be used as fiducial markers
for alignment of the tilted views. Single-axis tilt series (660 ˚ with 1 ˚
increments) were produced from samples using Xplore3D software and a
Tecnai-G2 (FEI Company, Eindhoven, Netherlands) electron microscope
operating at 200 kV and coupled to a 4k64k CCD camera. All 3D
reconstructions and subsequent 3D data analyses were performed using
the IMOD software package (Kremer et al., 1996). Tomogram generation
RESEARCH ARTICLE
by R-weighted back-projection was performed using ETOMO, followed
by joining of adjacent tomograms (of sections in a series) using the JOIN
window in IMOD. Virtual slices were manually segmented using
3DMOD, which was also used to produce 3D models.
Focused-ion-beam scanning electron microscopy
For observation by FIB-SEM, cells that had taken up HRP for 5 minutes
were processed for TEM as described above. The top of the resin block
containing the sample was trimmed to a pyramidal shape, and its surface
was smoothed by sectioning using a conventional diamond knife. The block
was then glued to an SEM stub using silver paint, with the surface of the
resin block pointing upwards, perpendicular to the microscope column.
Samples were imaged by using a Nova Nanolab 600 dual-beam microscope
(FEI Company) equipped with a gallium-ion source for focused-ion-beam
milling, and a field-emission gun and an in-lens secondary electron detector
for SEM imaging. Prior to milling and SEM imaging, the entire sample
surface was coated with a 3-nm-thick platinum layer using a gas injector
system located inside the main specimen chamber. The specimen stage was
tilted to 52˚ and exposed to the focused ion beam so that the plane of the
stage was parallel to the beam. A cross-sectional cut was introduced in two
stages. First, a coarse cut was made at high beam currents (typically 7–
20 nA) and at an accelerating voltage of 30 kV to create a trench that
enabled viewing of the cross-section. Usually, 50–150-mm-wide trenches
were cut into the specimen. In the second step, the ion beam was scanned
using a current of 3–7 nA to polish and smooth the surface. Back-scatter
electron images were recorded at accelerating voltages of 1–5 kV and a
beam current of 4000 pA in the immersion lens mode. To create a slice-
and-view image series, a step size of 25 nm was chosen for the removal of
material from the specimen surface with the focused ion beam. After image
capturing, the back-scattered electron images had their contrast inverted so
that they looked like conventional TEM images. Slice-and-view series were
automatically aligned using the XfAlign algorithm of IMOD, and then a
fine alignment was performed using MIDAS. All 3D models and
measurements were performed with 3DMOD. For statistical analyses of
cytopharynx length variations during the cell cycle, we used the GraphPad
Prism 6 software (GraphPad Software).
Acknowledgements
We are grateful for the technical assistance of Thiago Luis de Barros Moreira
(Centro Nacional de Bioimagem, Rio de Janeiro, Brazil) and Luis Sergio Júnior
(Inmetro, Rio de Janeiro, Brazil). We are most grateful to Flavia F. Moreira Leite
(Instituto de Biofı́sica Carlos Chagas Filho, Rio de Janeiro, Brazil) for critical
reading of the manuscript.
Competing interests
The authors declare no competing interests.
Author contributions
C.L.A. and J.C.V. designed and performed experiments. W.S. and N.L.C.S.
conceived and designed experiments and interpreted data. C.L.A. and N.L.C.S.
wrote the paper. All authors commented on drafts of the paper.
Funding
This work was supported by Conselho Nacional de Desenvolvimento Cientı́fico e
Tecnológico (CNPq) [grant number 472262/2012-2], Coordenação de
Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES) scholarships to C.L.A
and J.C.V., Financiadora de Estudos e Projetos (FINEP), Programas Núcleo de
Excelência (PRONEX) [grant number E-26/110.576/2010] and Cientista do Nosso
Estado [grant number E-26/102.850/2012] from the Fundação Carlos Chagas
Filho de Amparo à Pesquisa no Estado do Rio de Janeiro (FAPERJ).
Supplementary material
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.135491/-/DC1
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